Drug discovery method

ABSTRACT

A method of obtaining information about a chemically active area of a target molecule, for example for drug discovery, comprising: providing a set of substantially rigid chemical gauges; reacting said target with a plurality of gauges of said set of gauges, assaying a binding of said gauges with said target to obtain a plurality of assay results; and analyzing said assay results to obtain information about said chemically active area.

FIELD OF THE INVENTION

The present invention relates to methods of molecule affinitydetermination, for example, for use in discovering new drugs.

BACKGROUND OF THE INVENTION

The development of a new pharmaceutical, from conception to readinessfor marketing, typically costs hundreds of millions of dollars and takesmany years. The development process starts with a step of matching amolecule (a potential pharmaceutical) to a target, e.g., a protein in ahuman body or in a microorganism. The matching of a molecule to apharmaceutical is known as a drug lead, as it may lead to thedevelopment of a drug. The molecule is then modified to be more active,more selective and more pharmaceutically acceptable (e.g., less toxicand more easily administered). The failure rates at these stages arevery high.

With the development of combinatorial chemistry and automated screeningtechniques, a new method of drug discovery has been developed. In thisnew method, a large library of molecules is chemically tested against atarget, with the molecule having a best match being used as a startingpoint for finding a lead and/or as a lead. Some of these libraries areconstructed empirically, for example, based on available moleculesand/or molecules known to act as pharmaceuticals. Other libraries areconstructed to have a wide a range as possible of different molecules.Other libraries are constructed so that individual molecules will haveas great a chance as possible in matching a target. In general,molecules are selected to be as diverse as possible and to be drug like(e.g., size, chemical behavior) so that if a match is found it can serveas a lead.

Some references to such libraries and/or other discovery methodsinclude, Pickett S. D. at al., J. Chem. Inf. Comput. Sci. 36 (6), p.1214-23 (1996) and Ferguson A. M. et al., J. Biomol. Scr. 1 (2), p. 65(1996), Bunin A. B. et. al., Proc. Natl. Acad. Sci. USA 91, p. 4708-12(1994), Ellman J. et. al., Proc. Natl. Acad. Sci. USA 94, p. 2779-82(1997) and Maly D. J. et. al., Proc. Natl. Acad. Sci. USA 97 (6), p.2419-24 (2000), the disclosures of which are incorporated herein byreference.

Another, virtual, structure based, type of screening is known. In thevirtual method, a model of the target is generated (e.g., x-raycrystallography, estimated tertiary layout, analogy). Then, the affinityof a large number of molecules is determined by calculating dockingbehavior of a model of the molecule in the model of the target. Due tothe relatively primitive state of molecular modeling and the resultinglack of availability of models, this method is not currently verysuccessful.

Sunesis, inc., in D J Maly et al PNAS 97 (6), p 2419-24 (2000), thedisclosure of which is incorporated herein by reference, suggest usinglarge fragments of molecules as leads and then linking together suchmatching leads that are found into larger leads that are tested againfor matching. The fragments are provided with pre-defined linkers, forthe linking together.

PCT application PCT/US99/06734 (WO 99/49314), the disclosure of which isincorporated herein by reference, also describes a scheme of usingfragments, and then linking the fragments to provide leads.

SUMMARY OF THE INVENTION

An aspect of some embodiments of the invention relates to a targetcharacterization method, in which a plurality of small, measurementmolecules interact with a target and the target is characterized basedon an analysis of the interactions of the measurement molecules with thetarget. In an exemplary embodiment of the invention, none of themeasurement molecules is used as a lead or as a fragment of a lead, norare the molecules selected for interaction based on their drug-typediversity. Rather, the measurement molecules are selected based on theirexpected ability to measure various chemical and/or physical dimensionsof the target. In an exemplary embodiment of the invention, while thenumber of measurement molecules is relatively small (e.g., <10⁶), thisnumber spans the space of characterization of the target molecule andcan suffice to provide a relatively complete characterization of thetarget. In other embodiments, only a partial characterization is neededand/or obtained. Alternatively or additionally, while the measurementmolecules are selected for span reasons, they are also used as leads oras fragments of a lead.

In an exemplary embodiment of the invention, a complete process of drugdiscovery comprises:

-   -   (a) selecting a target;    -   (b) optionally selecting a set of measurement molecules useful        for the target, or using a universal library,    -   (c) characterizing the target using the set of measurement        molecules;    -   (d) reconstructing a pharmaceutical model of the target, based        on the characterization; and    -   (e) using the model to forward a discovery process, for example,        select, reject, filter and/or design a drug lead.

In some embodiments of the invention, a typical measurement molecule canmake one of several measurements, and a processing method, for exampleclustering, is optionally used to extract the particular measurementsmade by the molecules.

In an exemplary embodiment of the invention, the measurement moleculesare a set of chemical gauges, of which some, typically a small number,bind to the target, typically at one or more active sites of the target.The binding of a gauge to the target can be determined using variousassay methods, including substantially any of those known in the art,for example, by detecting a change in the chemical or biologicalbehavior of the target or by detecting a reduction in the number of freegauge molecules in a sample. In a particular example, a functional assayfor a protease (e.g., of an HIV protein) comprises linking a fluorescentmolecule onto a protein (or other peptide). The protease is allowed tointeract with a gauge, and this interaction is expected to reduce orcounteract (or enhance) its affinity for the protein, which change inaffinity may be determined by measuring the fluorescent properties(e.g., polarization) of the mixture of protein and protease. In anexemplary embodiment of the invention, each gauge is selected to have anaffinity to one or more particular geometric layouts. In an exemplaryembodiment of the invention, the total geometry of a target area isreconstructed from the determination of affinity (and/or lack ofaffinity) of a plurality of gauges.

In an exemplary embodiment of the invention, each of the gauges isconstructed from a scaffold to which a plurality of particular chemicalmoieties are attached. Three such moieties define a triangle of moietieswhich includes both a definition of the moieties at the vertexes and thedistance between the vertexes. In an exemplary embodiment of theinvention, the scaffolds and moieties are selected so that the trianglesare relatively rigid, however, some degree of play in the length of thetriangle sides (inter-moiety distances) may be desirable.

Each such moiety triangle matches a particular spatial layout of threebinding sites that match the moieties. Optionally, the distance betweenthe moieties is varied for different gauges, so that a range oftriangles with various desired combinations of moieties and distancesbetween the moieties is provided. As will be shown below, a gaugelibrary that includes a spanning set of such triangles, both withregards to distance and with regards to moiety is not prohibitivelylarge.

In an exemplary embodiment of the invention, the scaffold and/or themoieties are selected to have a minimum flexibility, so that they morespecifically define the geometric features that they match.

Optionally, the scaffolds and/or the moieties are selected to have a lowmolecular weight, so as to improve linking of low affinity gauges and/ortargets and possibly provide information for such cases.

In an exemplary embodiment of the invention, when selecting gauges for ameasurement library, some degree of overlap of moiety triangle isprovided. For example, an repetition overlap factor of 2 or 3 maybeprovided (e.g., each triangle appears in at least 2 or 3 gauges). Thisis expected to increase the probability of finding a triangle thatbinds, especially in view of problems which may occur such as stericclashes, chemical mismatch and/or solubility. Typically, an exactrepetition of the moiety triangle is not available, so a nearly similartriangle is used for providing the overlap. In some cases, the trianglesare selected so that for at least some pairs of moieties on the target,a triangle with a smaller distance between the same moieties and atriangle with a larger distance between the same moieties are bothavailable for binding. This provides a non-repetition overlap factor.Alternatively to 2 or 3, a lower or higher overlap factor, for example 4or 6, and/or possibly a fractional factor (e.g., an average overlap),may be used. The overlap may be uniform on the library, or a greateroverlap may be provided for some triangles and/or molecules, for examplefor molecules where there is a greater probability of steric clashingdue to the scaffold and/or other moieties, or based on experimentalresults which indicate that certain gauges and/or triangles aredifficult to bind.

It should be noted that if a molecule is required to distort in order tobind, its likelihood of binding is typically lower. Thus, the actualoverlap between two dissimilar triangles of two gauges may benon-uniform and dependent on the total binding probability. In general,if a probability of discovery of biding in an assay is negligible, it isassumed that the gauge does not bind. This helps define the range ofdistortion that can be used to define coverage and overlap. In someembodiments of the invention, the molecules are substantially rigid, sothe cut-off of degree of distortion is more clearly defined and limited.

A particular exemplary drug discovery process in accordance with anexemplary embodiment of the invention, is as follows:

(a) Synthesize a library of small molecules designed to span allpossible 3-point pharmacophores (all combinations of 3 elementarychemical moieties and distances between them). This is a finite librarywhich may include, for example ˜1100,000 compounds. This is termed a USL(Universal Screening Library), due to its generalized nature of ability(e.g., in some embodiments of the invention) to be used for mapping awide range of targets for which small molecule drugs are designed.

(b) For any target, screen the USL against that target, looking forweakly active compounds (affinity of ˜100 microM). Theoreticalconsiderations and experimental data indicate that 100-1000 hits shouldbe expected for any target.

(c) Computationally analyze the active molecules, seeking:

-   -   1. 3-Point-Pharmacophores (3PP's) involved in binding of the        hits.    -   2. Reconstruction of the binding-site topography in terms of        chemical moieties involved in binding. Generate the complete        pharmacophore (˜10-20 points) of the binding-site.

(d) Computationally identify molecules that may compliment a largeenough (e.g., 6-8 points for nanoMolar binding) subset of the fullpharmacophore. Optionally, by knowing which parts of these molecules arenot directly involved in binding, design them to meet predefineddrug-like qualities (e.g. using Lipinski's rules of 5).

(e) Using well known chemical knowledge, chose those molecules mostamenable to synthesis and other considerations (e.g., toxicity) andsynthesize those as possible drug candidates.

(f) Testing and iterations.

An aspect of some embodiments of the invention relates to estimating aspatial layout of binding locations in a target molecule. In anexemplary embodiment of the invention, the binding of a plurality ofsmall molecules to the target is determined, for example using assaymethods. In an exemplary embodiment of the invention, the smallmolecules are selected to have or are each modeled as a set ofgeometrical sub-structures which may, on its own, bind to the target. Inone example, the geometrical sub-structure may be three moietiesarranged in a triangle. In an exemplary embodiment of the invention, theassay results are analyzed to determine which of the many geometricalsub-structures in the small molecules, actually bind to the targetmolecule. In an exemplary embodiment of the invention, a clusteringmethod is used to determine which geometrical sub-structures bind, byclustering together molecules that bind and that have similargeometrical sub-structures. The output of the clustering method may be alist of all the probably binding sub-structures. Optionally, thesub-structures used for analysis and for design of the gauges istriangular.

In an exemplary embodiment of the invention, a score based method isused to convert a list of geometric sub-structures (e.g., triangles)into a complete geometric structure, by:

-   -   (a) generating possible structures from the list of        sub-structures;    -   (b) associating a “correctness” score with each structure; and    -   (c) selecting between structures based on their score.

In an exemplary embodiment of the invention, the score represents theprobability of two sub-structures sharing a portion in the structureand, optionally, a higher score is provided for a structure in which aportion is shared, as that represents a more cohesive structure.Alternatively or additionally, the score represents the probability oftwo different moieties binding to a same binding location, and,optionally, a higher score provided if more moieties share a samebinding site, as this represents a minimization of pharmacophore pointsto the minimum required. Other heuristic rules may be used as well.

In an exemplary embodiment of the invention, the set of all potentialmodels is not actually built. Instead a search is made of the space ofmodels and the models are built (and/or rejected) ad-hoc based on thedetermined sub-structures.

In an alternative embodiment of the invention, a clustering method isused, comprising for example:

-   -   (a) generating (all) possible structures from the found        triangles, optionally using particular construction rules;    -   (b) finding the most common large sub-structures that are shared        by multiple structures; and    -   (c) selecting a particular common sub-structure, optionally        using a scoring method, such as cluster size, edge size and        thresholding of cluster size, possibly selecting a most common        substructure from all those that pass a certain threshold. In        some cases, more than one final resulting sub-structure will be        provided.

It should be noted that an actual pharmacophore may not be a limitedsize and strictly defined entity, for example, a point that istechnically outside the active area, can act as a pharmacophore if asmall molecule drug binding to that point includes a tail that blocksthe active area from interacting with the substrate. Often however, the“relevance” of a binding area will decrease as the area is further awayfrom an active area, a control area and/or a conformance changing area.In addition, the binding affinity of a protein is often significantlysmaller away from such areas.

In an exemplary embodiment of the invention, the structures forclustering are generated in the following manner:

-   -   (a) a triangle is selected as a base sub-structure;    -   (b) a point is added to the base sub-structure, if there are two        triangles that, together with a triangle on the sub-structure,        define a tetrahedral; and    -   (c) (b) is repeated until there are no unused triangles left to        add.

An aspect of some embodiments of the invention relates to finding one ormore molecules (e.g., a drug lead) that is expected to match a target,from a plurality of geometric and/or chemical measurements of the targetarea. The measurements are optionally used to generate a reconstructionmodel of the target, against which model various processing methods maybe applied, for example using suitable computer hardware or software. Inan exemplary embodiment of the invention, the measurements are providedby interacting the target with a plurality of gauge molecules anddetermining the degree of binding of the gauge molecules to the target.For example, a set of triangular geometries is determined by gaugematches and is correlated to recreate a three-dimensional model of thetarget area.

Optionally, the target area is compared to structures of knownpharmaceuticals or pharmaceutical like materials, for example a druglead library. Alternatively or additionally, the target area geometry isused to select a most likely candidate from a relatively small pluralityof materials. Alternatively or additionally, the matching is used duringthe process of drug development, to select or reject modifications ofdrug leads, which do or do not match the target area geometry.

In a particular example, if one wants to satisfy Lipinsid's rules byadding or subtracting H-bond donors/acceptors, knowing which ones areimportant for binding would indicate which ones not to remove, andknowing which parts of the molecule are not important would indicatewhere additions can be made without hurting the binding.

An aspect of some embodiments of the invention relates to a library ofgauges for measuring a biochemical target. In an exemplary embodiment ofthe invention, the library comprises a large number of moleculesconstructed by attaching moieties on a relatively small number ofscaffolds. In an exemplary embodiment of the invention, the moieties areselected to have as low a molecular weight as possible. Alternatively oradditionally, the library is designed to cover, in a desired manner, aset of parametrically defined geometric sub-structures. Possibly, thegeometric sub-structures are triangles, with different moieties at theirvertexes. In one example, the range of different triangle dimensions isevenly covered.

In an exemplary embodiment of the invention, the library is selected toprovide same (overlapping) geometric sub-structures based on severalscaffolds and/or in several molecules, for example, each sub-structurebeing provided twice or thrice. Optionally, the overlapping is designedto take into account steric clashes and/or different chemistries ofdifferent, scaffolds and/or gauges.

In an exemplary embodiment of the invention, the scaffolds used includeat least two, at least five, at least seven, at least 10 or any greateror intermediate number, such as at least all of the following scaffolds:mono-carbone; pyrrole; quinoline pyrazinoquinazoline; isoindoloindole;isoindoloindole with an oxygen moiety attached; indolo[2,3-b]quinoline;pyrrolizine; 2,2′-bipyrrolone; indolizine; Thiophene; 1H-Pyrrole; Furan;Benzene; Pyridine; Pyrimidine; Pyrazine; 6H-Thieno[2,3-b]pyrrole;1,6-Dihydro-pyrrolo[2,3-b]pyrrole; 1H-Indole; Thieno[2,3-d]pyrimidine;6,7-Dihydro-pyrazolo[1,5-a]pyrimidine; Quinoline; Isoquinoline;Quinoxaline; 3,4-Dihydro-benzo[e][1,4]diazepin-5-one;3,8-Dihydro-4H-pyrrolo[2,3-e][1,4]diazepin-5-one;3,4-Dihydro-thieno[2,3-e][1,4]diazepin-5-one;3,6-Dihydro-4H-pyrrolo[3,2-e][1,4]diazepin-5-one;5H,11H-Dibenzo[b,f][1,5]diazocine-6,12-dione;1,4-Dihydro-10H-1,4,10-1,4,10-triaza-benzo[a]cyclopenta[e]cyclooctene-5,10-dione;4H,10H-1-Thia-4,10-diaza-benzo[a]cyclopenta[e]cyclooctene-5,11-dione;Dipyrrolo[1,2-c;2′,1′-e]imidazol-5-one;1,4,7,9-Tetrahydro-1,4,6,9-tetraaza-dicyclopenta[a,e]cyclooctene-5,10-dione;4,7,9-Trihydro-1-thia-4,6,9-triaza-dicyclopenta[a,e]cyclooctene-5,10-dione;2,4,9,Trihydro-1lambda*4*,6-dithia-4,9-diaza-dicyclopenta[a,e]cyclooctene-5,10-dione;6,9-Dihydro-5H-1-thia-5,8,9,triaza-cyclopenta[a]azulen-4-one;3,10,Dihydro-4H-[1,4]diazepino[5,6-b]indol-5-one;3,6-Dihydro-4H-[1,4]diazepino[6,5-b]indol-5-one;7,8-Dihydro-1H-1,7,10-triaza-cyclohepta[e]inden-6-one;8,9-Dihydro-3H-3,6,9-triaza-cyclohepta[e]inden-10-one;7,8-Dihydro-1H-1,5,8-triaza-cyclohepta[f]inden-9-one;8,9-Dihydro-5,6,9,11-tetraaza-cyclohept[b]naphthalene-10-one;3,4-Dihydro-[1,4]diazepino[5,6-b]quinolin-5-one;8,9-Dihydro-4,8,11-triaza-cyclohepta[a]naphthalene-7-one;11H-10,11-Diaza-benzo[b]fluorine; c-hydroxyacids; α-aminoacids; cohels;Bicyclo[2.2.2]octane; 2-Methylene-2,3-dihydrobenzo[1,4]dioxine;6,7-Dihydro-2H-pyrazino[1,2-a]pyramidine; 9H-Fluorene;1,4-Diaza-bictclo[2.2.2]octane; 1-Aza-bicyclo[2.2.2]octane;Pyrido[2,3-d]pyrimidine; 5-Methylene-1,5-dihydro-pyrrol-2-one;Bezno[4,5]imidazo[1,2-a]pyrimidine;1,4-Dihydro-benzo[4,5]imidazo[1,2-a]pyrimidine;4,10-Dihydro-1,4a,10-triaza-phenanthren-9-one;1,5-Dihydro-imidazo[1,2-a]pyrimidin-2-one;1,2,3,5-Tetrahydro-imidazo[1,2-a]pyrimidine;Thiazolo[3,2-a]thieno[2,3-d]pyrimidin-5-one;1,9-Dithia-4a,10-diaza-cyclopenta[b]fluoren-4-one;5,6-Dihydro-1-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-4-one;6,10-Dihydro-5H-1-thia-5,7,10a-triaza-benzo[e]azulen-4-one;4,5-Dihydro-3-thia-4,5a,10-triaza-cyclopenta[a]fluorine;8H-1-Thia-cyclopenta[a]indene;3-Thia-4,5a,10-triaza-cyclopenta[a]fluorine;6,7,9,11-Tetrahydro-10-thia-6,9-diaza-indeno[1,2-a]azulene-5,8-dione;2,3,6,7,12a-Hexahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione;5,10-Dihydro-4H-2,3a,10-triaza-cyclopenta[a]fluorine;5H-Pyrido[4,3-b]indole; 11H-Indolizino[1,2-b]quinolin-9-one;1,2-Dihydro-2,4a,9,-triaza-anthracene-3,10-dione;6H-Isoindolo[2,1-a]indole; 1,5-Dihydro-benzo[b][1,4]diazepin-2-one;5,10-Dihydro-dibenzo[b,e][1,4]diazepin-11-one;5,11-Dihydro-benzo[e]pyrido[3,2-b][1,4]diazepin-6-one;4,9-Dihydro-3-thia-4,9-diaza-benzo[f]azulen-10-one; Benzo[g]quinoxaline;Pyrazino[2,3-b]quinoxaline; Pyrido[2,1-b]quinazolin-11-one;1-Thia-4a,9-diaza-cyclopenta[b]naphthalene-4-one;2-Methylene-4H-benzo[1,4]thiazin-3-one.

In general, the greater the number of scaffolds, the easier it is tofind right sizes of gauges and also deal with a wider range of stericclash conditions and/or different chemistries. On the other hand,smaller number of scaffolds, promotes uniformity of chemical behaviorand synthesis methods.

In an exemplary embodiment of the invention, the moieties used include,at least 2, at least 4, at least 6, or any greater number, such as allof the following moieties: Me, Et, Pr, Ph, CO₂H, OH, NH₂, ketone,halides, such as Cl or Br, other acids such as SO₃H, PO₃H₂, andNH—C═NH(—NH₂) (Guanidine).

In general, using more moieties may provide greater accuracy incharacterizing binding, at a possible expense of library size. Usingfewer moieties may also simplify synthesis methods.

An aspect of some embodiments of the invention relates to selecting agauge library for use in characterizing a target. In an exemplaryembodiment of the invention, a range of dimensions of target geometriesis estimated, as well as bond types of binding locations. A set ofmolecules that spans the range of possible sizes and bond types isselected from a larger available set of molecules. The selection may be,for example, electronic with selected molecules being synthesized inresponse to selection or the selection is physical, with the gaugemolecules already available. Optionally, the estimation uses variousinformation known about the target. Alternatively or additionally, theestimation is made using a first screening library, that is, forexample, more flexible in the affinity of its bond types and/or usesmolecules that are more flexible.

Optionally, the gauges are selected so that the library will haveconsiderable repetition, for example to overcome steric clashes and/orother properties of the molecules, that might prevent binding.Optionally, the library includes at least one, or possibly more than onemulti-point binding geometries, for at least some of the physicalgeometries, for example, triangles and pentagons.

In accordance with exemplary embodiments of the invention, such alibrary can be used on its own or as part of a different library forvarious uses. In an exemplary embodiment of the invention, such aspanning library is used to increase the probability of binding of anyof the gauges in the library to the target, desirably, a considerablenumber of gauges. It is noted that a standard lead library oftenprovides no bindings at all. Optionally, the bindings results are usedto gather information about the target, especially statisticalinformation. Optionally, the statistical information is used to providestructural information about the target. Optionally, the structuralinformation comprises a chemical and/or geometrical structure of asignificant part of the target, for example, an active area thereof. Itshould be noted that in an exemplary embodiment of the invention, onceeven a single binding is found, useful information about the target isavailable and any library that assists in guaranteeing this binding hasa use.

An aspect of some embodiments of the invention relates to designingand/or creating a gauge library for use in characterizing targetmolecules by geometrical and/or chemical measurements.

In an exemplary embodiment of the invention, library constructioncomprises:

-   -   (a) identifying molecules that may be suitable as gauges;    -   (b) determining if the identified molecules provide required        gauges; and    -   (c) verifying that the molecules are realistic, for example        being readily synthesizable and/or having desirable chemical        behavior. It should be noted that this order is flexible, for        example as shown below.

In one example, this method is used when basing at least part of a gaugelibrary on existing libraries. In some libraries, (c) is alreadyperformed when the library is originally composed. Further, in somecase, rather than select molecules, known existing binding results ofcertain molecules are used as input, instead of selecting a gauge andphysically testing the binding affinity.

Alternatively, candidate gauges may be provided as a group, for example,when a new scaffold is added to a library. A large number of candidatesthen arise, as attachments of different moieties to the scaffold. Inthis case, however, an opposite step may be taken—a scaffold may berejected because it does not add any (or enough) gauges that do notoverlap with existing gauges. For some parts of the spanned space,scaffolds that generate few gauges may be suitable.

In an alternative method, chemical design methodology is applied todesign gauges and/or scaffolds that have desired properties and/orgeometries, for example, to fill in missing parts of a measurementspace.

In an exemplary embodiment of the invention, one or more of thefollowing are considered to be desirable properties of gauges, however,a gauge need not have all or even any of the following properties, inorder to be useful for some embodiments of the invention:

(a) High rigidity. This may allow measurements to be more exact,however, a small degree of flexibility may be desirable, to allowcomplete coverage of all the space. Rigid means that the length and/orrelative angles of the bonds do not change a significant amount.

(b) Low mass. This may increase the chance of bonding even if affinityis low and only three points on the gauge bind.

(c) Small size. This may allow targets to be more easily measured andsteric clashes more easily avoided.

(d) Non-toxic. This may allow the use of the gauge in living cells.However, due to the differing sensitivity of different cells, this oftencannot be ensured.

(e) Good chemical behavior. This means that the gauge is soluble andbinds under conditions that do not distort the gauge, or distort it by aknown amount.

(f) Strong binding. This means in one embodiment of the invention, forexample, 1-100 micromolar, which is useful for example if solubility islow or toxicity is high.

In an exemplary embodiment of the invention, one or more of thefollowing are considered to be good properties of scaffolds, however, ascaffold need not have all or even any of the following properties, inorder to be useful for some embodiments of the invention:

(a) Easy to attach moieties (e.g., synthesize gauges) and obtain puresolutions of particular gauges.

(b) Provide a wide range of sizes.

(c) Have many (e.g., ≧3, better >4, >5) attachment points. While everyhydrogen atom in a molecule is potentially an attachment point, in anexemplary embodiment of the invention, a useful attachment point isaccessible for chemical manipulation.

(d) What (relatively rare in other gauges) chemistries possibilitiesand/or gauge sizes are added to the library, by inclusion of thescaffold.

(e) Allow attachment of various combinations of moieties, as not allcombinations will work with all scaffolds.

In an exemplary embodiment of the invention, one or more of thefollowing are considered to be desirable properties of a gauge library:

(a) Spanning of a range of distances between bonds.

(b) Chemical spanning. At points on opposite ends of bonds, a wide rangeof moieties are provided.

(c) Sub-structure spanning. For the sub-structure selected, e.g., atriangle, all possible triangle configurations in a target can bind toat least one gauge in the library.

(d) Small. The smaller the library the better. For practical reasons,the library cannot be too small, however, very large libraries aregenerally not necessary.

(e) Variations of gauge properties within library to match the densityof gauge coverage, for example, less rigid bond lengths to cover missingor spaced apart bonds.

(f) Uniform coverage. Various types of uniformity may be provided, forexample, uniformity in absolute sizes or uniformity corrected forchemical dependencies. For example, the density of distances for shortbond lengths will be higher than for long bond lengths, to provide asame normalized density for different lengths.

(g) Degree and type of overlap. While more overlap is generally betterfor reconstruction and chemical generalization, it often comes at a costof library size and cost. An overlap of three (e.g., each triangle isprovided in three gauges) is an exemplary compromise.

In general, however, the desirable properties may depend on the target,environment and/or type of discovery method being applied. Inparticular, it is noted that in some cases, the generated library isonly partial, for example spanning only a part of the space, beingsuitable for only part of a target, being in a lower resolution, havingless (or no) overlap and/or being prone to fail for some types oftargets.

A broad aspect of some embodiments of the invention relates tomolecules, such as gauges and scaffolds and methods of synthesisthereof, which may find use for libraries in accordance with exemplaryembodiments of the invention.

There is thus provided in accordance with an exemplary embodiment of theinvention, a method of obtaining information about a chemically activearea of a target molecule, comprising:

-   -   providing a set of substantially rigid chemical gauges;    -   reacting said target with a plurality of gauges of said set of        gauges;    -   assaying a binding of said gauges with said target to obtain a        plurality of assay results; and    -   analyzing said assay results to obtain information about said        chemically active area. Optionally, said gauges allow rotation        of moieties of said gauges. Alternatively or additionally, said        gauges are constructed using a rigid scaffold.

In an exemplary embodiment of the invention, constituent atoms of saidgauges do not move more than 1 Å unless at least 20 Kcal/Mol are appliedto the gauge.

In an exemplary embodiment of the invention, analyzing comprisesidentifying a plurality of spatial and chemically specific bindingsconfigurations in said target active area. Optionally, saidconfigurations comprise triangular configurations. Alternatively oradditionally, identifying comprises identifying a configuration thatmatches a configuration of a bound gauge. Alternatively or additionally,identifying comprises identifying a configuration that does not match aconfiguration of a bound gauge. Optionally, identifying comprisesidentifying by statistical analysis of said assay results. Optionally,identifying comprises identifying by clustering.

In an exemplary embodiment of the invention, identifying comprisesassuming each gauge indicates a single configuration. Alternatively oradditionally, identifying comprises assuming at least some of the gaugesindicate a plurality of configurations. Alternatively or additionally,identifying comprises classifying gauges by chemical moieties atvertexes of said configurations.

In an exemplary embodiment of the invention, the method comprisesreconstructing a spatial map of at least part of said chemically activearea, from at least two of said assay results, said part including atleast four chemical binding areas. Optionally, said part includes atleast six chemical binding areas.

In an exemplary embodiment of the invention, the method comprisesreconstructing a spatial map of at least part of said chemically activearea, from at least two of configurations, said part including at leastfour chemical binding points. Optionally, said part includes at leastsix chemical binding areas.

In an exemplary embodiment of the invention, reconstructing comprises:

-   -   test-reconstructing a plurality of spatial maps from said        configurations;    -   scoring said maps; and    -   selected a spatial map based on its score. Alternatively or        additionally, reconstructing comprises:    -   test-reconstructing a plurality of spatial maps from said        configurations;    -   clustering said maps according to common substructures; and    -   selected a spatial map based on a relative property of a cluster        it belongs to. Optionally, said relative property comprises        size.

In an exemplary embodiment of the invention, said spatial map includesenough binding points to ensure binding of a small molecule drug havinga chemical profile matching the binding points. Optionally, said spatialmap includes at least 6 binding points. Optionally, said spatial mapincludes at least 8 binding points.

In an exemplary embodiment of the invention, said set of gaugescomprises a set of gauges with at least 10,000 gauges. Optionally, saidset of gauges comprises a set of gauges with at least 50,000 gauges.

In an exemplary embodiment of the invention, said gauges comprisemoieties arranged in spatial configurations and wherein said gauges areselected to span a virtual space of spatial chemical configurations.

In an exemplary embodiment of the invention, substantially each point ofvirtual space that is spanned by said gauges is covered by at least twogauges. Optionally, substantially each point of virtual space that isspanned by said gauges is covered by at least three gauges.

In an exemplary embodiment of the invention, at least 0.5% of saidgauges bind with said target. Optionally, at least 1% of said gaugesbind with said target. Optionally, at least 3% of said gauges bind withsaid target.

In an exemplary embodiment of the invention, at least 50% of said gaugesare defined by adding moieties to a set of fewer than 100 scaffolds.Optionally, at least 50% of said gauges are defined by adding moietiesto a set of fewer than 50 scaffolds.

In an exemplary embodiment of the invention, at least said set of gaugesuses fewer than 15 different chemical moieties to define the chemicalbehavior of said gauges.

In an exemplary embodiment of the invention, at least said set of gaugesuses fewer than 10 different chemical moieties to define the chemicalbehavior of said gauges.

In an exemplary embodiment of the invention, said assay is a functionalassay. Alternatively or additionally, said assay is a binding assay.Alternatively or additionally, said assay is a cellular assay.Alternatively or additionally, said assay is a flow-through assay.

In an exemplary embodiment of the invention, said functional assay isperformed in the presence of a natural substrate of said target.

In an exemplary embodiment of the invention, said target comprises aprotein including a biochemically active area adapted to engage asubstrate. Optionally, said chemically active area comprises an areaincluding said biochemically active area. Alternatively or additionally,said chemically active area comprises a control area of said protein.

In an exemplary embodiment of the invention, analyzing comprisesanalyzing successful binding of at least 60 gauges. Alternatively oradditionally, analyzing comprises analyzing successful binding of atleast 10 gauges. Alternatively or additionally, analyzing comprisesanalyzing successful binding of at least 100 gauges.

In an exemplary embodiment of the invention, identifying comprisesidentifying at least 40 different configurations. Alternatively oradditionally, identifying comprises identifying at least 10 differentconfigurations. Alternatively or additionally, identifying comprisesidentifying at least 100 different configurations.

In an exemplary embodiment of the invention, the method comprises:

-   -   comparing said map to a lead data base; and    -   selecting a lead from said data base for further use responsive        to a semblance or lack of semblance between said lead and said        map.

Alternatively or additionally, the method comprises:

-   -   comparing said map to a lead data base; and    -   rejecting a lead from said data base for further use responsive        to a semblance between said lead and said map.

Alternatively or additionally, the method comprises:

-   -   constructing a lead to have a semblance to said map. Optionally,        constructing comprises constructing using said gauges or        scaffolds used to define said gauges.

In an exemplary embodiment of the invention, the method comprises:

-   -   comparing said configurations to a lead data base; and    -   selecting a lead from said data base for further use responsive        to a matching of said configurations to said lead.

In an exemplary embodiment of the invention, the method comprisesconstructing a lead based on said configurations.

In an exemplary embodiment of the invention, the method comprisesselecting at least one of said gauges as a lead for drug discovery.

In an exemplary embodiment of the invention, the method comprisescomparing the binding of gauges with similar binding geometries toobtain steric clashing data; and

-   -   analyzing said steric clashing data to provide geometrical        information about said target.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of identifying the existence of a plurality ofchemical-spatial configurations in a target, comprising:

-   -   assaying the target with a plurality of gauges having know        chemical-spatial configurations at vertexes thereof, to provide        a plurality of assay results;    -   defining an array of spaces, one space for each set of chemical        behaviors of the vertexes of each configuration;    -   indicating said results according to said spaces, to generate        clusters; and    -   identifying the existence of a configuration in said target from        said clusters; Optionally, indicating comprises spreading an        indication responsive to a spreading function. Optionally, said        spreading function is dependent on an estimated energy of        binding of a gauge to said target.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of reconstructing a spatial shape of a chemicalbinding configuration of a target from a set of sub-shapes, each ofwhich indicates a part of said binding configuration, comprising:

-   -   selecting a base from said sub-shapes;    -   selecting at least two sub-shapes having the property that they        match each other at least along one side thereof and match said        base along another side thereof;    -   accumulating said sub-shapes to said base; and    -   repeating said selecting and said accumulating until all of said        sub-shapes are used or cannot be used, thereby providing a shape        of a binding configuration of said target. Optionally, the        method comprises variationally repeating said selecting,        accumulating and repeating using a different order of selection        of sub-shapes. Optionally, the method comprises repeating said        selecting a base and said variationally repeating for a        plurality of different base selections. Optionally, the method        comprises clustering a plurality of such shapes according to        shared sub-component shapes. Optionally, the method comprises        selecting a sub-component shape as a resulting shape based on        said clustering.

In an exemplary embodiment of the invention, said sub-shapes comprisetriangles. Alternatively or additionally, said sub-shapes definechemical behavior at their vertexes and wherein two sides are said tomatch if the chemical behavior at their vertexes match.

In an exemplary embodiment of the invention, two sides are said to matchif their length is similar.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of selecting a scaffold for use in generating a partof a screening library, comprising:

-   -   providing a potential scaffold molecule including a plurality of        possible attachment points for moieties;    -   determining a rigidity of the molecule; and    -   rejecting said potential scaffold molecule responsive to a lack        of rigidity of said scaffold. Optionally, said lack of rigidity        is absolute. Alternatively, said lack of rigidity is relative to        other potential scaffolds.

In an exemplary embodiment of the invention, the method comprisesselecting a scaffold based on a number of rings thereof.

In an exemplary embodiment of the invention, the method comprises:

-   -   determining a plurality of gauge molecules that can be generated        by adding moieties to said potential scaffold molecule;    -   determining for an existing library portion what spatial        chemical configurations are added by said molecules; and    -   selecting said potential scaffold molecule if one or more        significant spatial chemical configurations can be added by it        to said library portion. Optionally, the method: comprises        selecting a scaffold based on a number of configurations added        by said scaffold. Alternatively or additionally, said        significant spatial configurations are configurations not        previously provided or overlapped with,

There is also provided in accordance with an exemplary embodiment of theinvention, a method of selecting a gauge molecule to be added to ascreening library, comprising:

-   -   providing a set of chemical molecules and at least a part of a        screening library;    -   selecting a potential gauge molecule from said set of chemical        molecules; determining a rigidity of said potential gauge        molecule; and    -   rejecting said potential gauge molecule responsive to a lack of        rigidity of said gauge molecule. Optionally, said lack of        rigidity is absolute. Alternatively, said lack of rigidity is        relative to other potential scaffolds.

In an exemplary embodiment of the invention, the method comprises:

-   -   determining a spanning, in chemical configuration space, of said        part of a screening library;    -   determining at least one spatial chemical configuration of said        potential molecule; and    -   selecting said potential gauge molecule if it adds at least one        significant spatial chemical configuration to said screening        library.

Optionally, providing a set of molecules comprises generating saidmolecules using a single scaffold to which moieties are selectivelyattached. Alternatively or additionally, providing a set of moleculescomprises providing a chemical library.

In an exemplary embodiment of the invention, said gauge is selected ifit adds at least one spatial chemical configuration not previouslyprovided or overlapping a provided configuration.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of creating at least a portion of a screeninglibrary, comprising:

-   -   selecting a scaffold molecule to which moieties can be added;    -   determining a plurality of potential gauges which can be created        by attaching moieties to said scaffold; and    -   selecting a subset of said gauges that do not substantially        overlap in chemical configurations. Optionally, the method        comprises rejecting potential gauges that add over six spatial        chemical configurations.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of reducing a screening library, comprising:

-   -   for each molecule in at least part of said library, determining        substantially all the spatial chemical configurations of a        certain order of binding points provided by the molecule; and    -   removing a plurality of molecules which add redundant spatial        chemical configurations. Optionally, said certain order is        three.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of reducing a screening library, comprising:

-   -   for each molecule in at least part of said library, calculating        a binding probability of said molecules based on energetic        considerations; and    -   removing at least some molecules whose binding probability is        below a threshold value. Optionally, said binding probability is        calculated using a formula which is inversely dependent on a        flexibility of the molecule. Alternatively or additionally, said        binding probability is at least estimated based on a solubility        of the molecule.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of designing a screening library for a projectedtarget molecule task, comprising:

-   -   determining a desired range of distances between binding points        to be directly identified by said library;    -   determining a desired overlap between measures provided by gauge        molecules of said library;    -   determining a set of desired binding types to be discriminated        between; and    -   generating a plurality of gauges, said gauges each defining a        plurality of binding types and distances between them, such that        said gauges cover a spatial chemical configuration space that        includes said distances and said binding types with said desired        overlap. Optionally, generating a plurality of moieties        comprises generating by attaching moieties to scaffolds.        Alternatively or additionally, said gauges cover a spatial        chemical configuration space of triplets of binding points.        Alternatively or additionally, said projected target molecule        task comprises proteins.

In an exemplary embodiment of the invention, said overlap is at leasttwo. Alternatively said overlap is at least four. Alternatively, saidoverlap is at least six.

In an exemplary embodiment of the invention, said gauges aresubstantially rigid. Alternatively or additionally, said coverage takesinto account an inherent flexibility of binding.

In an exemplary embodiment of the invention, generating comprisesgenerating substantially same configurations by different gauges,thereby providing at least part of said overlap. Optionally, generatingcomprises providing a repetition factor of at least two.

In an exemplary embodiment of the invention, generating comprisesgenerating substantially different configurations by different gauges,which different configurations overlap due to a degree of flexibilitythereof, thereby providing at least part of said overlap.

In an exemplary embodiment of the invention, the method comprisesgenerating a set of drug leads for said target based on saidinformation. Optionally, the method comprises removing known drug leadsfor said target from said set.

There is also provided for in accordance with an exemplary embodiment ofthe invention, a lead set produced by one of the methods describedabove.

There is also provided in accordance with an exemplary embodiment of theinvention, a drug lead comprising:

-   -   a plurality of substantially rigid scaffolds molecule sections;    -   at least one link interconnecting said scaffold molecule        sections; and    -   a plurality of moieties attached to said scaffolds.

There is also provided in accordance with an exemplary embodiment of theinvention, a screening library comprising:

-   -   at least 10,000 molecules generated by attaching moieties to a        set of fewer than 50 scaffold molecules. Optionally, fewer than        20 scaffold molecules are used to generate said at least 10,000        molecules. Alternatively or additionally, said scaffolds include        at least one of the following scaffold molecules: Thiophene;        1H-Pyrrole; Furan; Benzene; Pyridine; Pyrimidine; Pyrazine;        6H-Thieno[2,3-b]pyrrole; 1,6-Dihydro-pyrrolo[2,3-b]pyrrole;        1H-Indole; Thieno[2,3-d]pyrimidine;        6,7-Dihydro-pyrazolo[1,5-a]pyrimidine; Quinoline; Isoquinoline;        Quinoxaline; 3,4-Dihydro-benzo[e][1,4]diazepin-5-one;        3,8-Dihydro-4H-pyrrolo[2,3-e][1,4]diazepin-5-one;        3,4-Dihydro-thieno[2,3-e][1,4]diazepin-5-one;        3,6-Dihydro-4H-pyrrolo[3,2-e][1,4]diazepin-5-one;        5H,11H-Dibenzo[b,f][1,5]diazocine-6,12-dione;        1,4-Dihydro-10H-1,4,10-1,4,10-triaza-benzo[a]cyclopenta[e]cyclooctene-5,11-dione;        4H,10H-1-Thia-4,10-diaza-benzo[a]cyclopenta[e]cyclooctene-5,11-dione;        Dipyrrolo[1,2-c;2′,1′-e]imidazol-5-one;        1,4,7,9-Tetrahydro-1,4,6,9-tetraaza-dicyclopenta[a,e]cyclooctene-5,10-dione;        4,7,9-Trihydro-1-thia-4,6,9-triaza-dicyclopenta[a,e]cyclooctene-5,10-dione;        2,4,9,Trihydro-1lambda*4*,6-dithia-4,9-diaza-dicyclopenta[a,e]cyclooctene-5,10-dione;        6,9-Dihydro-5H-1-thia-5,8,9,triaza-cyclopenta[a]azulen-4-one;        3,10,Dihydro-4H-[1,4]diazepino[5,6-b]indol-5-one;        3,6-Dihydro-4H-[1,4]diazepino[6,5-b]indol-5-one;        7,8-Dihydro-1H-1,7,10-triaza-cyclohepta[e]inden-6-one;        8,9-Dihydro-3H-3,6,9-triaza-cyclohepta[e]inden-10-one;        7,8-Dihydro-1H-1,5,8-triaza-cyclohepta[f]inden-9-one;        8,9-Dihydro-5,6,9,11-tetraaza-cyclohept[b]naphthalene-10-one;        3,4-Dihydro-[1,4]diazepino[5,6-b]quinolin-5-one;        8,9-Dihydro-4,8,11-triaza-cyclohepta[a]naphthalene-7-one;        11H-10,11-Diaza-benzo[b]fluorine; α-hydroxyacids; α-aminoacids;        cohels; Bicyclo[2.2.2]octane;        2-Methylene-2,3-dihydrobenzo[1,4]dioxine;        6,7-Dihydro-2H-pyrazino[1,2-a]pyramidine; 9H-Fluorene;        1,4-Diaza-bictclo[2.2.2]octane; 1-Aza-bicyclo[2.2.2]octane;        Pyrido[2,3-d]pyrimidine; 5-Methylene-1,5-dihydro-pyrrol-2-one;        Bezno[4,5]imidazo[1,2-a]pyrimidine;        1,4-Dihydro-benzo[4,5]imidazo[1,2-a]pyrimidine;        4,10-Dihydro-1,4a,10-triaza-phenanthren-9-one;        1,5-Dihydro-imidazo[1,2-a]pyrimidin-2-one;        1,2,3,5-Tetrahydro-imidazo[1,2-a]pyrimidine;        Thiazolo[3,2-a]thieno[2,3-d]pyrimidin-5-one;        1,9-Dithia-4a,10-diaza-cyclopenta[b]fluoren-4-one;        5,6-Dihydro-1-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-4-one;        6,10-Dihydro-5H-1-thia-5,7,10a-triaza-benzo[e]azulen-4-one;        4,5-Dihydro-3-thia-4,5a,10-triaza-cyclopenta[a]fluorine;        8H-1-Thia-cyclopenta[a]indene;        3-Thia-4,5a,10-triaza-cyclopenta[a]fluorine;        6,7,9,11-Tetrahydro-10-thia-6,9-diaza-indeno[1,2-a]azulene-5,8-dione;        2,3,6,7,12a-Hexahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione;        5,10-Dihydro-4H-2,3a,10-triaza-cyclopenta[a]fluorine;        5H-Pyrido[4,3-b]indole; 11H-Indolizino[1,2-b]quinolin-9-one;        1,2-Dihydro-2,4a,9,-triaza-anthracene-3,10-dione;        6H-Isoindolo[2,1-a]indole;        1,5-Dihydro-benzo[b][1,4]diazepin-2-one;        5,10-Dihydro-dibenzo[b,e][1,4]diazepin-11-one;        5,11-Dihydro-benzo[e]pyrido[3,2-b][1,4]diazepin-6-one;        4,9-Dihydro-3-thia-4,9-diaza-benzo[f]azulen-10-one;        Benzo[g]quinoxaline; Pyrazino[2,3-b]quinoxaline;        Pyrido[2,1-b]quinazolin-11-one;        1-Thia-4a,9-diaza-cyclopenta[b]naphthalene-4-one;        2-Methylene-4H-benzo[1,4]thiazin-3-one.

In an exemplary embodiment of the invention, at least 4 of saidscaffolds have exactly a single ring. Alternatively or additionally, atleast 4 of said scaffolds have exactly two rings. Alternatively oradditionally, at least 4 of said scaffolds have exactly three rings.Alternatively or additionally, at least 4 of said scaffolds have exactlyfour rings. Alternatively or additionally, said library includes atleast 50,000 thus generated molecules. Alternatively or additionally,said library includes at least 100,000 thus generated molecules.

In an exemplary embodiment of the invention, said scaffolds include atleast three of said following scaffold molecules. Alternatively oradditionally, said scaffolds include at least ten of said followingscaffold molecules.

In an exemplary embodiment of the invention, said generated moleculesare substantially rigid. Alternatively or additionally, said moleculesspan a configuration space of spatial geometrical patterns of bindingpoint types, including at least 25% of the patterns that exist inprotein targets. Optionally, said molecules span at least 50% of thepatterns.

In an exemplary embodiment of the invention, said molecules span a spacedefining at least 4 distinct binding point chemistry types.

In an exemplary embodiment of the invention, said molecules span a spacedefining at least 5 distinct binding point chemistry types.

There is also provided in accordance with an exemplary embodiment of theinvention, a screening library, comprising:

-   -   at least 100 gauge molecules generated by attaching moieties to        at least one of the following scaffolds: Thiophene; 1H-Pyrrole;        Furan; Benzene; Pyridine; Pyrimidine; Pyrazine;        6H-Thieno[2,3-b]pyrrole; 1,6-Dihydro-pyrrolo[2,3-b]pyrrole;        1H-Indole; Thieno[2,3-d]pyrimidine;        6,7-Dihydro-pyrazolo[1,5-a]pyrimidine; Quinoline; Isoquinoline;        Quinoxaline; 3,4-Dihydro-benzo[e][1,4]diazepin-5-one;        3,8-Dihydro-4H-pyrrolo[2,3-e][1,4]diazepin-5-one;        3,4-Dihydro-thieno[2,3-e][1,4]diazepin-5-one;        3,6-Dihydro-4H-pyrrolo[3,2-e][1,4]diazepin-5-one;        5H,11H-Dibenzo[b,f][1,5]diazocine-6,12-dione;        1,4-Dihydro-10H-1,4,10-1,4,10-triaza-        benzo[a]cyclopenta[e]cyclooctene-5,11-dione;        4H,10H-1-Thia-4,10-diaza-benzo[a]cyclopenta[e]cyclooctene-5,11-dione;        Dipyrrolo[1,2-c;2′,1′-e]imidazol-5-one;        1,4,7,9-Tetrahydro-1,4,6,9-tetraaza-dicyclopenta[a,e]cyclooctene-5,10-dione;        4,7,9-Trihydro-1-thia-4,6,9-triaza-dicyclopenta[a,e]cyclooctene-5,10-dione;        2,4,9,Trihydro-1lambda*4*,6-dithia-4,9-diaza-dicyclopenta[a,e]cyclooctene-5,10-dione;        6,9-Dihydro-5H-1-thia-5,8,9,triaza-cyclopenta[a]azulen-4-one;        3,10,Dihydro-4H-[1,4]diazepino[5,6-b]indol-5-one;        3,6-Dihydro-4H-[1,4]diazepino[6,5-b]indol-5-one;        7,8-Dihydro-1H-1,7,10-triaza-cyclohepta[e]inden-6-one;        8,9-Dihydro-3H-3,6,9-triaza-cyclohepta[e]inden-10-one;        7,8-Dihydro-1H-1,5,8-triaza-cyclohepta[f]inden-9-one;        8,9-Dihydro-5,6,9,11-tetraaza-cyclohept[b]naphthalene-10-one;        3,4-Dihydro-[1,4]diazepino[5,6-b]quinolin-5-one;        8,9-Dihydro-4,8,11-triaza-cyclohepta[a]naphthalene-7-one;        1H-10,11-Diaza-benzo[b]fluorine; α-hydroxyacids; α-aminoacids;        cohels; Bicyclo[2.2.2]octane;        2-Methylene-2,3-dihydrobenzo[1,4]dioxine;        6,7-Dihydro-2H-pyrazino[1,2-a]pyramidine; 9H-Fluorene;        1,4-Diaza-bictclo[2.2.2]octane; 1-Aza-bicyclo[2.2.2]octane;        Pyrido[2,3-d]pyrimidine; 5-Methylene-1,5-dihydro-pyrrol-2-one;        Bezno[4,5]imidazo[1,2-a]pyrimidine;        1,4-Dihydro-benzo[4,5]imidazo[1,2-a]pyrimidine;        4,10-Dihydro-1,4a,10-triaza-phenanthren-9-one;        1,5-Dihydro-imidazo[1,2-a]pyrimidin-2-one;        1,2,3,5-Tetrahydro-imidazo[1,2-a]pyrimidine;        Thiazolo[3,2-a]thieno[2,3-d]pyrimidin-5-one;        1,9-Dithia-4a,10-diaza-cyclopenta[b]fluoren-4-one;        5,6-Dihydro-1-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-4-one;        6,10-Dihydro-5H-1-thia-5,7,10a-triaza-benzo[e]azulen-4-one;        4,5-Dihydro-3-thia-4,5a,10-triaza-cyclopenta[a]fluorine;        8H-1-Thia-cyclopenta[a]indene;        3-Thia-4,5a,10-triaza-cyclopenta[a]fluorine;        6,7,9,11-Tetrahydro-10-thia-6,9-diaza-indeno[1,2-a]azulene-5,8-dione;        2,3,6,7,12a-Hexahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione;        5,10-Dihydro-4H-2,3a,10-triaza-cyclopenta[a]fluorine;        5H-Pyrido[4,3-b]indole; 11H-Indolizino[1,2-b]quinolin-9-one;        1,2-Dihydro-2,4a,9,-triaza-anthracene-3,10-dione;        6H-Isoindolo[2,1-a]indole;        1,5-Dihydro-benzo[b][1,4]diazepin-2-one;        5,10-Dihydro-dibenzo[b,e][1,4]diazepin-11-one;        5,11-Dihydro-benzo[e]pyrido[3,2-b][1,4]diazepin-6-one;        4,9-Dihydro-3-thia-4,9-diaza-benzo[f]azulen-10-one;        Benzo[g]quinoxaline; Pyrazino[2,3-b]quinoxaline;        Pyrido[2,1-b]quinazolin-11-one;        1-Thia-4a,9-diaza-cyclopenta[b]naphthalene-4-one;        2-Methylene-4H-benzo[1,4]thiazin-3-one.

Optionally, said molecules are generated using at least one of thefollowing scaffolds: Thiophene; 1H-Pyrrole; Furan; Benzene; Pyridine;Pyrimidine; Pyrazine; 6H-Thieno[2,3-b]pyrrole;1,6-Dihydro-pyrrolo[2,3-b]pyrrole; 1H-Indole; Thieno[2,3-d]pyrimidine;6,7-Dihydro-pyrazolo[1,5-a]pyrimidine; Quinoline; Isoquinoline;Quinoxaline; 3,4-Dihydro-benzo[e][1,4]diazepin-5-one;3,8-Dihydro-4H-pyrrolo[2,3-e]([1,4]diazepin-5-one;3,4-Dihydro-thieno[2,3-e][1,4]diazepin-5-one;3,6-Dihydro-4H-pyrrolo[3,2-e][1,4]diazepin-5-one;5H,11H-Dibenzo[b,f][1,5]diazocine-6,12-dione;1,4-Dihydro-10H-1,4,10-1,4,10-triaza-benzo[a]cyclopenta[e]cyclooctene-5,11-dione;4H,10H-1-Thia-4,10-diaza-benzo[a]cyclopenta[e]cyclooctene-5,11-dione;Dipyrrolo[1,2-c;2′,1′-e]imidazol-5-one.

In an exemplary embodiment of the invention, said at least 100 moleculescomprise at least 300 molecules. Alternatively or additionally, said atleast 100 molecules of said library are generated using a single one ofsaid scaffolds.

There is also provided in accordance with an exemplary embodiment of theinvention, a screening library comprising a set of at least 10,000substantially rigid molecules. Optionally, said set comprises at least50,000 substantially rigid molecules. Alternatively or additionally,said set comprises at least 100,000 substantially rigid molecules.

In an exemplary embodiment of the invention, said set is selected tohave a an expected binding rate of at least 0.1% of the library forprotein targets in general. Optionally, said expected binding rate is atleast 0.5%.

In an exemplary embodiment of the invention, said set is designed toprovide molecules with a uniformity of hit probability for a generalizedtarget of within a ratio of 1:100 for the whole set. Optionally, saidratio is within 1:10.

In an exemplary embodiment of the invention, said set spans a space ofspatial chemical configurations, each such configuration defining acertain plurality of binding points having distances between them, theset covering substantially all possible configurations in the space in agiven range of distances.

There is also provided in accordance with an exemplary embodiment of theinvention, a screening library, comprising:

-   -   a plurality of at least 5,000 gauge molecules, each such        molecule defining at least one spatial configuration of binding        type points,    -   wherein substantially each point in a space of such        configurations is covered by at least two different gauge        molecules. Optionally, each point is covered by at least two        substantially identical spatial configurations. Alternatively or        additionally, each point is covered by at least two        substantially different spatial configurations. Alternatively or        additionally, said space is a space of triangles defined by        binding type at vertexes and distances between vertexes.        Optionally, said space includes distances of between 4 Å and 8 Å        (angstrom=10⁻¹⁰ meters). Alternatively or additionally, said        space includes distances of between 2 Å and 10 Å. Alternatively        or additionally, said space includes at least 5 different        binding types. Optionally, said space includes at least 7        different binding types.

In an exemplary embodiment of the invention, said space includesomni-directional binding types. Alternatively or additionally, saidspace includes directional binding types.

In an exemplary embodiment of the invention, said substantially eachpoint in said space is covered by at least three gauges.

In an exemplary embodiment of the invention, substantially all thegauges include a plurality of configurations of said space.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of obtaining information about a binding behavior ofa target molecule, comprising:

-   -   providing a set of substantially rigid chemical gauges, a        significant number of said gauges being expected to bind with        said target;    -   reacting said target with a plurality of gauges of said set of        gauges; and    -   physically analyzing a structure of said target bound to a        gauge. Optionally, physically analyzing comprises analyzing        using NMR. Alternatively or additionally, physically analyzing        comprises analyzing using X-ray crystallography. Alternatively        or additionally, physically analyzing comprises analyzing using        binding with a set of gauges. Alternatively or additionally, the        method comprises virtually super-imposing a plurality of        structures obtained by said physically analyzing.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of constructing a lead, comprising:

-   -   providing a set of substantially rigid chemical gauges;    -   reacting said target with a plurality of gauges of said set of        gauges;    -   assaying a binding of said gauges with said target to obtain a        plurality of assay results; and    -   constructing a lead based on said assay results. Optionally,        constructing a lead comprises linking together a plurality of        gauges found to bind in said assaying. Alternatively or        additionally, constructing a lead comprises modifying an        existing molecule to have moieties that correspond to binding        locations found by said assaying.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the invention will be described withreference to the following description of exemplary embodiments, inconjunction with the figures. The figures are generally not shown toscale and any measurements are only meant to be exemplary and notnecessarily limiting. In the figures, identical structures, elements orparts which appear in more than one figure are preferably labeled with asame or similar number in all the figures in which they appear, inwhich:

FIG. 1 is a schematic diagram of a target protein including a pluralityof binding points;

FIG. 2 is a flowchart of a method of drug discovery, in accordance withan exemplary embodiment of the invention;

FIG. 3 is a flowchart of a method of target measurement, in accordancewith an exemplary embodiment of the invention;

FIG. 4A is a schematic illustration of an exemplary gauge, in accordancewith an exemplary embodiment of the invention;

FIG. 4B shows the gauge of FIG. 4A, interacting with the target proteinof FIG. 1;

FIG. 5 is a flowchart of a method of determining which triangles didbind to a target, in accordance with an exemplary embodiment of theinvention;

FIG. 6A is a flowchart of a method of determining a spatial layout ofbinding locations from the results of the method of FIG. 5, inaccordance with an exemplary embodiment of the invention; and

FIG. 6B is a flowchart of an alternative method of determining a spatiallayout of binding locations from the results of the method of FIG. 5, inaccordance with an exemplary embodiment of the invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

-   1. OVERVIEW-   2. EXEMPLARY PROCESS OF DRUG DISCOVERY-   3. DETAILS OF PROCESS    -   3.1 TARGET MEASUREMENT-   4. EXEMPLARY ASSAYS    -   4.1 FUNCTIONAL ASSAY    -   4.2 BINDING ASSAY-   5. GAUGES, GENERAL    -   5.1 EXEMPLARY GAUGE    -   5.2 NUMBER OF MOIETIES IN A MEASURE    -   5.3 NUMBER OF MOIETIES IN A GAUGE    -   5.4 MOIETY TYPES    -   5.5 OVERLAP OF MEASURES IN A SET-   6. RECONSTRUCTION    -   6.1 TRIANGLE EXTRACTION    -   6.2 LAYOUT CONFIGURATION RECONSTRUCTION    -   6.3 RECONSTRUCTION VARIATIONS    -   6.4 ALTERNATIVE RECONSTRUCTION METHOD-   7. ANALYSIS    -   7.1 OVERVIEW    -   7.2 RECONSTRUCTION VERIFICATION    -   7.3 BINDING STRENGTH    -   7.4 INTER-BOND INTERACTIONS    -   7.5 GEOMETRIC ANALYSIS    -   7.6 DETERMINATION OF STERIC CLASHES    -   7.7 IDENTIFICATION. OF CONTROL AREAS    -   7.8 OTHER MAP ANALYSIS-   8. USE IN DRUG DISCOVERY PROCESSES    -   8.1 OVERVIEW    -   8.2 DRUG GENERATION    -   8.3 LEAD GENERATION    -   8.4 LEAD DESCRIPTION    -   8.5 LEAD SEARCH    -   8.6 LEAD REJECTION    -   8.7 TARGETED MAPPING    -   8.8 TARGET SUITABILITY TESTING    -   8.9 TARGET PARTITIONING    -   8.10 DRUG AND LEAD ANALYSIS AND ENHANCEMENT    -   8.11 DRUG SELECTION    -   8.12 DRUG ENHANCEMENT    -   8.13 DRUG FAILURE ANALYSIS AND REENGINEERING    -   8.14 ADDITIONAL DRUG DISCOVERY RELATED ANALYSIS    -   8.15 STREAMLINE DISCOVERY PROCESS    -   8.16 UTILITY GENERATION-   9. EXEMPLARY DISCOVERY APPLICATIONS    -   9.1 OVERVIEW    -   9.2 SCREENING BASED DRUG DESIGN    -   9.3 ALTERNATIVE SCREENING BASED DRUG DESIGN    -   9.4 STRUCTURE-BASED DRUG DESIGN    -   9.5 MODULAR ASSEMBLY OF LIGANDS-   10. EXEMPLARY NON-DISCOVERY USES-   11. USING PRIOR INFORMATION-   12. ITERATIVE MEASUREMENT-   13. GAUGES, PHYSICAL PROPERTIES    -   13.1 OVERVIEW    -   13.2 SCAFFOLD    -   13.3 VOLUMETRIC GEOMETRY OF GAUGES    -   13.4 FLEXIBILITY    -   13.5 GAUGE LENGTHS    -   13.6 ENVIRONMENTAL STABILITY    -   13.7 UNIQUENESS OF GAUGES AND OVERLAP OF SIDES AND TRIANGLES    -   13.8 GAUGE MASS AND SIZE-   14. PARTICULAR AND GENERAL GAUGE SET DESIGN    -   14.1 EXAMPLE SPANNING LIBRARY SIZE    -   14.2 GAUGE SUBSET SELECTION    -   14.3 GAUGE LIBRARY DESIGN    -   14.4 LIBRARY BUILDING METHOD    -   14.5 SCAFFOLD SELECTION METHOD    -   14.6 GAUGE SELECTION METHOD    -   14.7 GAUGE SYNTHESIS    -   14.8 MIXED LIBRARY DESIGN    -   14.9 ENSURING LIBRARY RELIABILITY    -   14.10 HUMAN INTERACTION DURING LIBRARY DESIGN-   15. EXPERIMENTS AND EXAMPLES    -   15.1 EXPERIMENT 1    -   15.2 EXPERIMENT 2-   16. SYNTHESIS BOOK    -   16.1 Benzenes, Pyrimidines 6-membered ring scaffold    -   16.2 lndolo[2,3-b]quinoline 6,6,5,6 cyclic scaffold    -   16.3 isoindoloindoles and isoindoloindolones 6,5,5,6 tetra        cyclic scaffolds        -   16.3.1 Isoindoloindolones    -   16.4 The single atom scaffold    -   16.5 Benzodiazepines 6,7 bicyclic scaffold    -   16.6 Pyrazinoquinazolinone-6,6,6 tricyclic scaffold    -   16.7 Pyrrole-5 membered ring scaffold    -   16.8 Thiophenes and related scaffolds        -   16.8.1 5,5 bicyclic scaffolds        -   16.8.2 5,6-bicyclic scaffolds        -   16.8.3 5,8,5 5,8,6 tricyclic and 5,5,8,6 5,5,8,5 tetracyclic            scaffolds        -   16.8.4 5,7 bicyclic scaffold        -   16.8.5 5,6,5,6 Tetracyclic and 5,6,5 tricyclic scaffolds        -   16.8.6 5-6-5-6 tetracyclic scaffold        -   16.8.7 5-6-5 tricyclic scaffold            1. Overview

The high specificity of many biological molecules, such as enzymes, iscreated by the existence, in such a molecule, of a particular spatialarrangement of binding locations. It is believed that for a substratemolecule to succeed in usefully interacting with the enzyme, it mustmatch (at least part of) the particular spatial arrangement. In thepharmaceutical industry, this specificity can be utilized by findingsmall molecules that mimic the shape and chemical affinities of thesubstrate molecule. In a typical drug discovery method, such a smallmolecule is found by trying out millions of small molecules and, oncefinding a molecule which appears to have some affinity, chemically finetuning that “lead” until a better binding is found. In an exemplaryembodiment of the invention, the particular spatial arrangement ismapped and this map is used to assist in the drug discovery process and,ultimately, in finding new and useful small molecule drugs. It should benoted that, in general, the spatial geometry of the binding locations isthree dimensional.

In the following description, the molecule is called a target and thespatial arrangement is called a target area or a pharmacophore. However,as will be clear, a mapping method in accordance with an exemplaryembodiment of the invention and/or its derivatives have uses beyond drugdiscovery, for example, developing herbicides and targeted anti-bodies.Thus, the terms used are used for convenience and not for limiting thedesired coverage, except where noted otherwise.

FIG. 1 is a schematic diagram of a target protein 100 including aplurality of binding locations 102 (and 108). As shown, bindinglocations 102 are arranged in a target area 104, which is designed toaccept the substrate of the protein. In some proteins, a target area ofinterest is a control area 106 of the protein (with binding locations108), which, when bound, changes the behavior of the protein (e.g.,changing the configuration of the substrate receiving area of theprotein). Possibly, a plurality of non-functional binding locations 110are found on the outside of the protein.

Although the following description focuses on finding small moleculesfor affecting enzymatic proteins, target 100 may be any bio-moleculewhose biological behavior may be desirably affected by the binding of amolecule to it. For example, target 100 may be one or more of DNA, RNA,signaling proteins such as hormones, structural hormones, growthfactors, other proteins, anti-bodies, cell receptors, ion channels,cytokines, complexes, membranes, toxins (biological and synthetic),small and large molecule drugs and carbohydrates. Non-biologicalapplication are also envisioned, for example for assessing enzymes usedfor washing and industrial uses. In addition, the searched-for moleculeneed not be a small molecule, for some applications, for example, it maybe a peptide, protein, antibody or metal complex.

In accordance with some exemplary embodiments of the invention, themapping of target area 104 (or 106) is provided by making multiplegeometrical and/or chemical affinity measurements of the target area andthen correlating the measurements to provide a three dimensional modelof target area 104. In an exemplary embodiment of the invention, themeasurements are made using a set of selective gauge molecules. In anexemplary embodiment of the invention, the gauges are selective tocertain bond geometries and/or certain chemical affinities, with anoptional small range of flexibility. In a set of gauges a large range ofgeometries, sizes and/or affinities is optionally provided using alarger number of specific gauges.

In an exemplary embodiment of the invention, each gauge molecule makesmultiple measurements simultaneously and there is an overlap between themeasurements made by different gauge molecules. A processing step isoptionally provided in which the composite measurement from gauges areinter-related to yield an indication of individual measurements whichare then used for reconstructing a three-dimensional map. Additionalside information is optionally used for the processing and/or or foranalyzing and/or using the results of the processing. Various examplesof such side information are described below.

2. Exemplary Process of Drug Discovery

FIG. 2 is a flowchart of a method of drug discovery 200, in accordancewith an exemplary embodiment of the invention. At 202, a target 100 forwhich a drug is to be developed, is provided. Optionally, at 204, asubset of gauges is selected for the measurement of target 100.Alternatively, a single set of gauges is used for all targets.

At 206, the gauges are used to measure the spatial layout of interactionlocations 102 and/or 108.

At 208, a model of at least part of the active and/or control areas oftarget 100 is reconstructed from the measurements. At 210 and 212 one ormore molecules that match the measurements are determined. At 214, thematching molecules are further processed to provide drugs.

Further details of this method are described below. Alternative methodsare also described below.

3. Details of Process

3.1 Target Measurement

FIG. 3 is a flowchart of a method of target measurement 300, inaccordance with an exemplary embodiment of the invention. At 302, anamount of target 100 and one or more gauges are combined in a container,and possibly allowed to incubate (304) so that gauges can bind tointeraction locations in target 100. In some embodiments of theinvention, the target is also incubated with a substrate or anothermolecule. Such incubation may be provided for various reasons, forexample, to force a conformal change on the target to assist indissolving, to keep the target alive and/or as part of a functionalassay. The target may be in a relatively pure state, for example apurified replicated DNA segment. Alternatively, the target may beprovided in a more natural environment, for example in a living cell orwith associated molecules (e.g., whose interactive effects may beunknown). Optionally, a plurality of overlapping gauges (i.e.,overlapping in them being able to measure same or similar spatialgeometries) are incubated together in a same assay.

At 306, the degree of binding of the gauge to target 100 is optionallydetermined. The method used may depend on the type of assay used,various examples of which are provided below. Alternatively oradditionally, at 308, an effect on the function of target 100 isdetermined, various examples are provided below.

It should be noted that assays suitable for detecting binding of a testmolecule to a target molecule are well known for drug discovery and manyare suitable for the present invention, possibly with no modification.

The assaying process may then be repeated with a different gauge and/ordifferent conditions (310), such as solvent, temperature and pH. Varyingthe conditions may be used, for example, to determine the strength ofthe binding and/or to compensate for unavailable gauges, for example byforcing conformal changes on target 100. The repetition may depend onpreliminary binding results for one or more gauges and/or may depend onpreliminary measurements or measurement failures.

In an exemplary embodiment of the invention, the assays are at a 1-100micro Molar concentration of the gauge. However, other concentrationsmay be used. The concentration may depend, for example, on thesolubility of the gauge and/or various toxic or other effects associatedwith the gauge. In many cases, the concentrations used will depend onthe sensitivity of the assay.

The purity of the target may or may not be important, for example,depending on the affinity of the gauge to the impurities and/or on thesensitivity of the assay to the impurities.

4. Exemplary Assays

4.1 Functional Assay

Many types of functional assaying methods are known in the art. Ingeneral, the treated target is provided with its normal substrate (forproteins) and a measurement of enzymatic activity is used to determinethe functional effect of the gauge, relative to a baseline or a controlportion of material. Automated parallel assay devices, such asmanufactured by Tecan (Switzerland), Zymark (USA) or Cybio (DE) canperform multiple functional assays in parallel, for example, fordifferent gauges and/or for better statistics on a single gauge-targetmatch.

Functional assays may be on various levels, for example, on a molecular,cellular or organism level. In general, any known functional assay maybe used to assay the functionality of a gauge.

In an exemplary embodiment of the invention, the gauge acts like aligand of the target and compete or otherwise affects the functionalityof the target. These effects may be of various types, for example, thegauge may bind where the normal substrate is supposed to bind, the gaugemay bind near where the substrate binds, but still block the substratefrom binding, the gauge may bind in a way that does not block thesubstrate but would, if the gauge were larger (suitable for a bindingassay) and/or the gauge may be agnostic rather than antagonistic in itsbehavior, enhancing the affinity of the target for the substrate.

DNA targets can be assayed, for example, using replication methods(e.g., to see if replication is inhibited or enhanced). Alternatively,DNA targets are assayed by determining their interaction with DNA chipsafter the test binding. Such DNA chips typically include a substrate onwhich a plurality of short DNA segments are mounted in a known pattern,with the segments selected to bind (e.g. be specific and complementary)to portions of a searched for DNA sequence and/or match sections of anon-linear DNA segment. It is expected that the type and/or relativefrequencies of bindings to various short DNA segments on a DNA chipdepend on the degree and/or location of binding of a gauge to a DNAmolecule. For example, a gauge may block a certain part of a DNAmolecule from matching up with a DNA chip segment. In another example, agauge may force a conformal change in a DNA molecule, which change willinterfere with binding with one DNA chip segment but which may allowbinding with a previously unsuitable DNA chip segment.

4.2 Binding Assay

In a binding assay, the binding of a gauge to a target is directlymeasured. It should be noted, however, that a binding assay may be lessindicative than a functional assay, as a gauge can bind at a locationoutside of the target area and provide no useful information about thetarget area. In addition, the sensitivity of a binding assay may belower, since the detection sensitivity of binding is usually lower andtypical binding rates are also quite low. However, in some cases, afunctional assay cannot be performed, for example if the gauge interactswith the substrate, or if a target function is not known, or may bedifficult or time consuming to perform, for example if the assayrequires a living cell. Also, a gauge may bind in an active area withoutthis binding affecting the functionality, as measured by a particularfunctional assay.

Various types of binding assays are known in the art and may be used,for example as described in the Handbook of Drug Screening, edited byRamakrishna Seethala and Prabhavathi B. Fernandes, in Drugs and thePharmaceutical Sciences, Volume 114, New York, N.Y., Marcel Dekker,2001, the disclosure of which is incorporated herein by reference.

Both functional assays and binding assays may be performed in many ways,the current technology being robotic performance of tests and theemerging technology being flow-through analysis (e.g., using DNA chips).It should be noted that 100,000 test systems are becoming available,which means that in some embodiments of the invention, screening using agauge library can be completed in one step (day). Optionally, this isused to prevent the need to clean out gauge delivery systems betweenscreening targets.

In some embodiments of the invention, the binding assay (of a functionalassay) includes modifying a gauge, for example, attaching a fluorescentmaterial to the gauge. Depending on the attachment point, this may causeconformal changes in some of the gauges and/or cause steric clashes. Itis expected that the overlap between gauges will overcome this problem,at least in a significant number of cases.

In other embodiments of the invention, the gauges are not changed, orare changed in non-material ways. For example, for an NMR binding assayor an x-ray crystallography binding assay no change is required. In aradioactivity based assay, radioactive isotopes can be used in thegauges. In an exemplary embodiment of the invention, non-radioactiveisotopes (half spin isotopes) are used in producing the gauges, toprovide binding detection and/or better analysis of NMR data. In theseassays, unbound gauges may be separated from the targets, for example,using methods known in the art, for example, if the target is bound to asurface, washing will remove unbound gauges.

In some embodiments of the invention, the binding of the gauge has anon-functional effect on the target, which may be detected or measured,for example, affecting a vibration frequency of a fluorescent tailattached to the gauge or the target. In an exemplary embodiment of theinvention, the gauge binds with the target in a manner similar to thatof a ligand of the target. Various techniques, for example as known inthe art (e.g., NMR, IR) may be used to analyze the combined target/gaugestructure. Optionally, once a binding gauge or other substrate is found,a gauge set is used to measure the combined target/ligand structure.

In some binding assays, a plurality of differently marked gauges may beassayed simultaneously and possibly differentially, for example, by aattaching a different florescent marker to different gauge used togetherand/or using different radioactive isotopes for different gauges.

Optionally, the binding assay (and/or a functional assay) may includechanging various environmental parameters, such as temperature, pHand/or other environmental variables, for example to determine astrength of binding.

In an exemplary embodiment of the invention, a binding assay is used todetermine a baseline level of binding of the gauge outside active areasof the target. In one example, the degree of binding of a particulargauge to alpha helixes in a protein may be known from an analog of thetarget. The total binding to the target, however, includes bindings tonon-helix parts of the protein and/or target areas of the target.

In an exemplary embodiment of the invention, it is noted that a largenumber of hits are expected and/or an overlap between gauges isprovided. As a result, lower quality and/or faster assays are used,since noise caused by low binding rates may be less of a problem. In oneexample, borderline results form two assays are combined, based on arepetition of triangular measures between the gauges used in the assays.

5. Gauges, General

5.1 Exemplary Gauge

FIG. 4A is a schematic illustration of an exemplary gauge 400, inaccordance with an embodiment of the invention.

Gauge 400 comprises a scaffold 402 and four chemical moieties 406, 410,414 and 422 attached to scaffold 402 via bonds 404, 408, 412 and 420,respectively. This is only an exemplary gauge, as the properties of allof these elements may vary, for example as described below. Inparticular, one or more of the type of moiety, number of moieties, typeof bond, distance between moiety and scaffold, type of scaffold andlocation of connection to the scaffold may be varied for differentgauges, sets of gauges and/or embodiments of the invention.

In an exemplary embodiment of the invention, a plurality of moietiescooperate to define a measure. In an exemplary embodiment of theinvention, the gauge purpose is to detect interaction locations thatbind to those moieties that define a measure at the distances betweenthe moieties. The matching of a measure to the target molecule may beindicated by the binding of the gauge. In an exemplary embodiment of theinvention, a basic unit of measure is a triangle (or other geometricshape) defined by a subset of all the moieties. As will be describedbelow, the shape of a triangle has particular properties which make itsuitable for some embodiments. In general, if a gauge includes more thanthe number of moieties in a measure (e.g., more than two moieties for alinear measure, more than three for a triangle), more than one measuremay be provided by a single gauge. Thus, in the exemplary embodiment ofthe invention shown, a plurality of different triangle measures aredefined in a single gauge. In some embodiments and in some cases, agauge will include only one measure, for example, gauge 400 includesonly a single four-point measure, but four triangle measures. Exemplarymethods of determining which of various possible measures actuallybound, are described below.

One triangle geometry is shown by dashed lines 416, 418 and 420 thatdefine the distances between pairs of moieties of gauge 400. As notedabove, in an exemplary embodiment of the invention, the gauge purpose isto detect interaction locations that bind to those moieties (406, 410,414) at the distances defined by dashed lines 416, 418 and 420 (e.g.,triangle sides). Assuming gauge 400 included only moieties 406, 410 and414, then a binding of gauge 400 to target 100 can be is used as anindication that three interaction sites, of a type suitable to bind tomoieties 406, 410 and 414 are approximately at the respective distancesdefined by gauge 400. Since gauge 400 defines multiple triangles, abinding of gauge 400 indicates that at least one of the trianglesdefined by the moieties, binds.

FIG. 4B shows gauge 400, interacting with target 100, at threeinteraction locations 450, 452 and 454. Non-interacting moieties and therest of the gauge are not shown.

5.2 Number of Moieties in a Measure

As noted, each plurality of moieties defines a measures. While thepresent invention accommodates, in some embodiments thereof measures,with two, three, four and/or other numbers of moieties and/or gauge setsincluding a mixture of different measures, in an exemplary embodiment ofthe invention, the basic measure used is a triangle, with threemoieties. Using a triangle may provide one or more of the followingpotential benefits:

(a) A triangle defines a stable spatial relationship, which may beuseful as a unit component when “constructing” a model in threedimensions of the target area, from the binding results.

(b) There are fewer possible triangles than four-sided measures (forexample). Thus, generating a library that includes measures that coveran entire space is less time consuming. Further, as it is desirable insome embodiments of the invention to provide overlap between measures,such overlapping measures are more easily provided if there are fewermeasures. It is possible that chemical limitations may prevent theconstruction of high-order measure gauge libraries.

(c) A triangle always lies in a plane (e.g., three points define aplane), which may be mathematically useful for some reconstructionmethods.

(d) For some applications, a triangle represents the lowest number ofbinding points that will result in a measurable binding to a targetactive area. A typical drug includes six or more binding points, oftenas many as ten or more. Conversely, a higher-order measure may bind toostrongly. In other applications, the optimal number of moieties in ameasure may be higher or lower, of course.

Alternatively, a measure including two moieties are used, for example,defining lines. Alternatively or additionally, four- or higher valancemeasures are used, for example, to define more uniquely an interactionlocation configuration. In some embodiments of the invention, a mix ofdifferent valance measures may be used, in the gauge set and/or in thereconstruction, for example, 2-point, 3-point, 4-point and 5-pointmeasures, which may or may not be planar.

5.3 Number of Moieties in a Gauge

In an exemplary embodiment of the invention, the number of moieties in agauge is between four and ten, however, a smaller (e.g., three) orgreater number may be provided. Some scaffolds may be limited in thenumber of different moieties, moiety positions and/or moietiescombinations possible. Larger numbers of moieties are generallydesirable if the moieties define different triangle measures.Conversely, gauges with multiple attachment points and/or gauges withmany moieties may be more prone to steric clashes and/or other adverseinteractions between the moieties, which inhibit binding.

While the scaffold itself has chemical properties and may be consideredas having moieties, in some embodiments of the invention, theseproperties are ignored, for example during library design and/or duringbinding results analysis. Alternatively, the properties of the scaffoldmay be considered, for example only during analysis and/or duringlibrary design.

5.4 Moiety Types

In an exemplary embodiment of the invention, the moieties are selectedto reflect the types of bonds that the drug is expected to make with thetarget. In an exemplary embodiment of the invention, the moieties areselected based on their chemical behavior. If a particular behavior isexhibited by several moieties, in an exemplary embodiment of theinvention, only a smallest one of the moieties is selected. In someembodiments of the invention, multi-purpose moieties, which can bind toseveral different binding sites, are used instead of moieties which canonly bind to one type of target site. The specificity of the moietiesselected may depend, for example, on the total number of moieties, theirsize and their amenability for chemical processing. It should be notedthat some of the moieties are directional, while others arenon-directional. Where available, non-directional bonds may be preferredover directional bonds. In some exemplary embodiments of the invention,two levels of measurement are performed, a coarse resolution level and afine resolution level. More specific moieties may be used during thefine resolution level of measurement. Additional details and methods foroptionally reducing the number of moieties used in some embodiments ofthe invention, are described below.

Following is a list of moieties of which one or more may be attached togauges:

-   -   a. Hydrogen bond donor. Directional bond.    -   b. Hydrogen bond acceptor. Directional bond    -   c. Positive charge. Non-directional bond.    -   d. Negative charge. Non-directional bond.    -   e. Aromatic ring. Directional bond    -   f. Hydrophobic group. Non-directional in general, however, some,        e.g., rings, may be directional with a preferred direction        perpendicular to the ring plane.        Different moieties may be used in other embodiments of the        invention, for example, also providing one or more of Halogen,        Carbonyl, Phosphate and Sulfate bonds. It should be noted that        the different moieties may differ greatly in the their chemical        affinities or they may differ less or even slightly. In some        exemplary gauge sets, the slight difference between moiety        affinities is used to fine tune a measurement distinction        between bond types.

With respect to the directional bonds, in some embodiments of theinvention, it is assumed that the bond has sufficient spatialflexibility so that a small number, e.g., seven, different directionalbonds will suffice to cover all the possible bond directions.Alternatively, smaller or greater numbers of bond directions may beused. Optionally, different directional bonds have different numbers ofdirections represented in a gauge library. The angular distribution ofthe directions may be, for example uniform, or it may be non-uniform,for example depending on the bond type.

Several different sizes of hydrophobic bonds may exist. In an exemplaryembodiment of the invention, two sizes are selected and represented bydifferent moieties. An aromatic ring may also serve, as an oversizedhydrophobic moiety. Alternatively or additionally, an aromatic ring isused to match aromatic bonds with other rings and/or some types ofhydrogen bonds.

The above selection of moieties and directions results in 25 uniquemoieties, which can be attached to scaffolds. An exemplary set ofmoieties is described below.

In an exemplary embodiment of the invention, a subset of the abovemoieties is used. Use is made of the rotational flexibility of hydrogenbond donors and/or receivers. Although such flexibility will generallyreduce chemical bonding probability, the mass of a hydrogen atom used ina hydrogen bond moiety is sufficiently low that the reduction inprobability may not materially affect the results of the measurementmethod, at least for some gauges and assays.

Alternatively or additionally, rotational flexibility is allowed foraromatic rings. Although aromatic rings have a high mass, the large bondarea of the ring compensates for the reduction in bond strength causedby allowing rotational flexibility of the ring.

Alternatively or additionally, some polar bonds may be represented by asingle moiety, such as OH, which can act as both a hydrogen bond donorand as an acceptor.

Optionally, for example if chemical information can be done without,more general moieties are used and a smaller number of triangles in alibrary is spanning.

5.5 Overlap of Measures in a Set

In an exemplary embodiment of the invention, the triangle space as awhole is spanned by providing a plurality of triangles, each withsufficient freedom in its parameters (e.g., bond length, chemicalaffinity), so that each triangular arrangement of binding points can beexpected to bind to one of the triangles to a measurable degree.Optionally, the coverage of each triangle in the triangle space overlapswith the coverage of other triangles, to ensure that no parts of thespace are left uncovered.

As will be explained in greater detail below, in an exemplary embodimentof the invention, a gauge library is designed such that each possibletriangular arrangement of binding points appears in (or fits within theparameters of) more than one gauge. In some cases, exactly congruenttriangles cannot be provided, instead, triangles that are roughlycongruent are provided (e.g., similar moieties, side lengths). Thesecongruent triangles may have the same coverage in triangle space or not.For example, assuming same moieties, two triangles with the followingside lengths are provided: (3, 4, 5) and (3.1, 3.9, 5.2) (measurementsin angstrom. These triangles may, for example, cover the part oftriangle space from (2, 3, 4) to (4, 5, 6).

In some embodiments of the invention, at least some of the trianglespace is spanned by a set of triangles with overlapping coverage. Forexample, for the same part of triangle space, the provided triangles are(2, 3, 4.5) and (2.5, 3.5, 5.3), which have overlapping, but differentcoverage.

While overlapping is useful for various reasons, for example, asdescribed below, it does increase the size of the library. Whenoverlapping is provided, the reconstruction method used optionally takesthe overlapping into account.

6. Reconstruction

After process 300 (FIG. 3) is repeated for as many gauges as desired,the measured affinities of gauges 400 to target 100 are optionally usedto reconstruct a model of the spatial distribution of interaction areas102. An exemplary method is described below.

In an exemplary (theoretical) mapping process for a particular targetmolecule, which uses a 75,000 gauge library, it is expected that about400 of the gauges will bind to the target. Due to repetition oftriangles in the library and/or due to the overlap in coverage ofnon-congruent triangles in the exemplary library, the number of realtriangles defined by the target area and bound to by gauges is expectedto be smaller. In one (theoretical) example, the number of “real”triangles that are defined by the target area and bound to by gauges is100 different triangles.

Taking for example a 10-point pharmacophore, such a pharmacophore mayinclude, for example, 10*9*8/6 triangles, which is 120 triangles. Insome embodiments of the invention, not all of these triangles areidentified, for example, due to high similarity between triangles (belowdistinguishing ability) or due to lack of binding (e.g., due to stericclashes). The 10 point structure can, of course be reconstructed withfewer than 100% of the triangles, especially of the missing trianglesare missing randomly. For example, 50% of the triangles may besufficient.

However, the actual situation is more forgiving. A typical pharmacophoremay include 20 points, of which, typically only between 8 and 10 need tobe identified in order to provide good binding. Thus, any substructureof the pharmacophore that includes 8-10 correct points can serve as agood starting point for drug generation. Fewer identified points canalso be useful, for example as described below.

Although various methods may be used to reconstruct the layout, in anexemplary embodiment of the invention a two step method is used. First,the “real” triangles are estimated from the results of the assay,optionally using a clustering algorithm. Then, a suitable layout usingthe triangles is found, optionally using a scoring based searchalgorithm or a clustering algorithm. In other implementations, a singlestep or multiple step method may be used.

6.1 Triangle Extraction

In an exemplary embodiment of the invention, this step of the processhas two parts, however, in other implementations, this step has a singlepart or more than two parts. One part is determining which trianglemeasures matched. This part may be less than trivial, for example, dueto the fact that each gauge includes multiple triangles. However, therepetition of triangles between gauges may assist in differentiation.Another, optional, part of the process is determining the real distancesinvolved, rather than those defined by a measure. For example, a realdistance between two moieties may be 4.3 angstrom, while bindingtriangle measures have distances of 4 and 5 angstrom. In someembodiments of the invention, it is desirable to estimate the realdistance, 4.3 angstrom, from the binding results. Optionally, this isprovided by the overlap in coverage of the different triangle measures.

In an exemplary embodiment of the invention, the two parts of theprocess are provided in a single compound process, for example usingclustering. Alternatively a two step method may be used. Optionally, aniterative method is used with an estimate of which measures bound beingused to estimate real distances and the real distances being used toimprove the earlier estimate of which measures bound.

FIG. 5 is a flowchart of a method 500 of determining which triangles didbind to a target, in accordance with an exemplary embodiment of theinvention.

At 502, a space is defined for each type of triangle (defined by themoieties of the triangle). Each such space has three dimensions, eachone representing a length of a side of the triangle.

At 504, a notation is made in a space at a location {x,y,z} if a gaugeincluding that type of triangle with sides of lengths {x,y,z} was shownto bind to the target. It should be noted that for two differentscaffolds, exactly matching triangles may be difficult to generate.Instead, the triangles may be nearly matching, for example havingslightly different lengths of sides.

In an exemplary embodiment of the invention, the assay results are usedas a binary input, there is either a bond or not. Alternatively, forexample if conformal changes are observed or there is a measure ofactivity and/or bonding, the bond strength may be represented by acontinuous or multi-step amplitude, using a hit notation.

In an exemplary embodiment of the invention, if a single gauge includesmultiple triangles, a hit is marked in each one of the relevant spaces.Alternatively or additionally, if a single triangle can match twodifferent type triangles, for example due to overlap between moietyaffinity, it is also marked in multiple spaces. Optionally, theamplitude of the marking is normalized to the number of spaces that aremarked by the gauge. Alternatively or additionally, a differentamplitude is provided in each space, responsive to an a prioriprobability of bonding.

At 506, the hits notations are replaced by a spatial spread function. Inan exemplary embodiment of the invention, the spread function representsthe probability of that triangle forming a bond at different distancesrepresented by the spread. Alternatively or additionally, the spreadingis between spaces, for example, if two moieties overlap in theiraffinities.

Alternatively, the hit indication is provided originally as a spreadingfunction.

In an exemplary embodiment of the invention, the spreading is a definedas $f = {\mathbb{e}}^{- \frac{\Delta\quad x^{2}}{\sigma\quad x^{2}}}$where Δx is the difference between the lengths of the sides and σx is avalue representing the difficulty in bending the molecule so that it canperform the bond. In an exemplary embodiment of the invention, σx is afunction of x, for example σx=α{square root}{square root over (x)}. Inan exemplary application, parameter “a” is 1.414. Possibly, the spreadfunction is non-uniform in space, for example, to reflect non-uniformcharacteristics of the bond. Optionally, at least some of the spreadingfunctions are derived empirically, by binding gauges having controlleddistances between bonds, with targets having known models. Alternativelyor additionally, such empirical testing is used for other purposes, forexample, to determine flexibility in bond length, multiple chemicalaffinity of moieties and/or symmetry of the spreading function.Optionally, targets are classified according to their flexibility aswell. Optionally, in an iterative process, once a model is estimated, aflexibility of the target is estimated and/or decided, for example forma table, and used to correct the spreading function used.

The spread hits are then combined, for example by addition, and peaksare found in the result (508). In an exemplary embodiment of theinvention, peaks are selected based on their shape. Alternatively oradditionally, peaks are selected based on their amplitude passing athreshold. This threshold can represent, for example, the number oftriangles that need to bind, to indicate a possible match. The thresholdmay be the same for all spaces or it may be different. Optionally, thethreshold and/or decision making method is selected based on theclustering statistics, for example from a table of previous empiricalresults. Alternatively or additionally, the threshold is selected sothat a minimum number of matches be found. Optionally, if there is alarge number of sub-threshold matches, a different gauge set is used forthe binding process. It is noted that in some embodiments of theinvention, for any given triplet of binding points there are generallyabout 12 triangles, or more, that can be expected to bind. For example,both a shorter side and a longer side are expected to bond to a pair ofbinding locations having an intermediate distance between them. Inaddition, each triangle type can appear multiple times, for example,three times in the set. In some sets, each (or some) triangle point inthe triangle space is covered by 24 triangles−8 triangle designs thathave longer and shorter sides in various combinations, times 3, if eachtriangle is provided three times. Additional overlap may be provided byambiguous moieties.

Optionally, by analyzing correlation between spaces and gauges, somefour-point geometrical matching (or higher) may be found as well.

6.2 Layout Configuration Reconstruction

FIG. 6A is a flowchart of a method 600 of determining a spatial layoutof binding locations from the results of the method of FIG. 5, inaccordance with an exemplary embodiment of the invention. In anexemplary embodiment of the invention, the method comprises constructingall the configurations (e.g., three dimensional shapes) that can beconstructed from the identified triangles and ranking the configurationsusing a scoring method, ultimately selecting the configuration with ahighest score.

At 602, all the possible configurations that can be constructed from thetriangles found in FIG. 5, are constructed. Alternatively to buildingcomputer models of all the possible configurations, in an exemplaryembodiment of the invention, the configurations are generated ad hoc.For example, in conjunction with the scoring method described below, aconfiguration may be constructed, or its construction advanced, only ifit is likely to have a useful score. For example, once a configurationsolution has a score below the highest found so far, that lower solutionis ignored.

In an exemplary embodiment of the invention, the construction method isby building up a structure piece by piece. For example, a triangle isadded to an existing configuration only if has a side length and/ormoieties that match a side length with a pair of moieties on thestructure. A threshold of size difference may be defined for allowingthe matching of two sides. Alternatively or additionally, a threshold ofmatching between moieties may be defined. Optionally, the moieties arerequired to match at the ends of the matching side, or to have anoverlapping chemical behavior. Such thresholds may depend on the lengthand/or types of moieties and/or other properties of the gauges and/orthe target. It is noted that a first gauge may bind to a particularbinding location using a different binding method from a second gauge,as long as the binding location supports both binding methods.

In an exemplary embodiment of the invention, the construction of aconfiguration is by sequentially selecting a triangle from the list ofavailable (bond) triangles, until all the triangles are used at leastonce. Used triangles may remain in the list for repeated use.Alternatively, the configuration may be built up using modules, each ofwhich is constructed from sub-modules, and, ultimately, triangles.

At 604, a score is calculated for each configuration. Such a score isoptionally a heuristic value indicating the reasonableness of the assayresults being derived from the target having the configuration. Variousscoring methods may be used. In an exemplary embodiment of theinvention, the scoring method is based on the particular linkingtogether of triangles in the configuration and/or on the probability ofthe triangles themselves being correct in the first place.

In an exemplary embodiment of the invention, the score is a product ofscores for each shared triangle side. In an exemplary embodiment of theinvention, the score for a triangle side that is shared between twotriangles is an estimated probability of the two sides of the twotriangles binding to a same pair of binding locations. In an exemplaryembodiment of the invention, the score is the product of the abovespreading function, for the x, y and z axes. Alternatively oradditionally, other, simpler scores, may be used, for example, basedonly on the difference in sizes of the sides.

In an exemplary embodiment of the invention, the score does not dependon the lack of a triangle. For example, if a generated configurationincludes a three point configuration for which no suitable gaugematched, it is not assumed that the configuration is incorrect, nor isthe score reduced. Alternatively, the score may be reduced responsive tothe existence of triangles that are found in a configuration and notfound on any matching gauge, for example, based on their count.

Alternatively or additionally, some configurations may be ruled outbased on heuristics, for example rules that describe what the layouttypically looks like. Alternatively or additionally, prior informationis used to rule out some configurations, for example, a partial model orknowledge of a molecule that binds well to the target.

At 606, the structure with the highest score is selected as the maplayout of the binding locations for the target. As noted above, 602-606may be carried out as an iterative search and construction method, forexample with structures being built ad hoc as the search progresses andindicates a certain structure has a score above a threshold (and so willits dependents). Many suitable search methods are known in the art, forexample, in the art of graph search and in the art of searching gametrees (e.g., for chess playing programs).

6.3 Reconstruction Variations

In an exemplary embodiment of the invention, a target may have severalactive areas. In an exemplary embodiment of the invention, thereconstruction is allowed to recreate a disjoint configurationstructure, with each disjoint part representing a map of one targetarea. Optionally, such a reconstruction may be required even for asingle active area, if enough triangles (e.g. gauge moieties) thatinterconnect the disjoint parts failed to bind (for various reasons)and/or were not available in the gauge set used, so that a continuousstructure cannot be reconstructed from the triangles that did match.

Optionally, the above reconstruction allows a triangle to appear onlyonce in a reconstructed configuration. Even if a triangle actuallyappears twice (or more) in the real configuration, the redundancy ofsimilar triangles will generally still enable the structure to bereconstructed. Alternatively or additionally, a triangle is allowed toappear more than once, however, this may affect the score, for example,reducing it. Alternatively, an iterative experimental approach, asdescribed below, is used, to block part of the target (e.g., with asuitable antibody or small molecule drug) and see if the triangle stillmatches.

Optionally, user intervention is allowed, for example, for viewing thefinal structure or several candidate structures. For example, if adetermination cannot be made, a human may be requested to select amongoptions, force certain matches and/or configuration parts and/or toremove certain possibilities from consideration, based on, for examplehuman experience and judgment and/or additional information about thetarget of various types.

It should be noted that one possibly output of the clustering and/orshape reconstruction methods is an input to an interactive processand/or to further drug development. For example, the application of theabove methods can show where more exact data is lacking for forming acomplete result and/or where there are ambiguities between possiblesolutions.

It should be noted that the resulting structure may have a mirror (e.g.,symmetry) ambiguity, due to the sole use of triangles. Optionally, thisambiguity is solved by using at least one 4- or higher-point measure,optionally constructed or selected to bind in only one of thepossibilities. Alternatively or additionally, the effect of stericclashes is used to distinguish between the two possibilities.Alternatively or additionally, prior information is used to distinguishbetween them.

6.4 Alternative Reconstruction Method

FIG. 6B is a flowchart 620 of an alternative reconstruction method,using clustering for shape reconstruction, in accordance with anexemplary embodiment of the invention.

At 622, a triangle is selected from the set of found triangles, thatwere found to bind in the assay and clustering of FIG. 5. This triangleis used as a base for constructing a structure.

At 624, a pair of triangles is selected from the remaining foundtriangles, such that the two triangles share a side with each other andeach triangle also shares a side with a part of the structure (e.g.,which two sides of the structure may or may not be sides of a sametriangle, depending, for example on the implementation). When thetriangle pair is added to the structure the structure grows by one pointin space. 624 is repeated (626) until no triangle pairs can be added.This completes one potential structure.

Often, there are several possible choices to make at 624, for example,for selecting the triangle pair and/or for deciding where to add them.At 628, a tree of possible structures is performed, by repeating 624 and626 for each possible choice of triangle pairs and their location. Thisprocess may be done, a priori, for example, by spawning multiple threadseach time multiple triangle pairs are available for selection and/oreach time such pairs may be attached at different locations.

At 630, 622-628 are repeated by selecting all possible triangles asbases, in turn (or in parallel). Alternatively, other methods ofgenerating all the possible structures from the triangles may be used.Optionally, a pruning method is used, for example, if a structure isclearly unsuitable or unable to utilize a significant percentage of thetriangles (e.g., 30%, 50%, 70% or any smaller, intermediate or greatervalue), the structure is dropped. Generally, the greater the number oftriangles allowed to be ignored, the easier it will be to provide astructure (e.g., even under noisy conditions). However, the structurewill be less constrained by the assay results and may be lessdependable.

At 632, all substructures found in the generated potential structures.Optionally, only some of the substructures are found, for example, onlythe largest or only those above a certain size. In an exemplaryembodiment of the invention, the method applied is a maximum likelihoodalgorithm for finding a most likely structure.

At 634, these substructures are clustered, with each point representinga structure in which the substructure is found. In an exemplaryembodiment of the invention, the clustering space is defined pertriangle type (e.g., type of moieties on the triangle) and the space isspanned by the sides of the triangles. Thus, for example, a 10 pointsub-structure of a 20 point structure is marked in a space that includesthe same number of moiety types as the sub-structure, with a location inthat space determined by the three Cartesian locations of each of thepoints (e.g., 30 dimensions for a 10 point sub-structure). Variousorientations are optionally dealt with by selecting a certain triangleto be a base triangle having an orientation. Alternatively oradditionally, the space is marked with structures in a rotationallysymmetric manner (or thus analyzed) so that the results from differentorientations may be compared. An exemplary algorithm is described in R.Nussinov, H. J. Wolfson, “Efficient Detection of Three DimensionalStructural Motifs in Biological Macromolecules by Computer VisionTechniques”, PNAS, volume 88, pp. 10495-10499, December 1991, thedisclosure of which is incorporated herein by reference.

At 636, a best substructure is selected. It is assumed that if asubstructure is common enough and large enough it is both correct anduseful. In an exemplary embodiment of the invention, a thresholding isapplied to select only those substructures with structures and clustersover a minimum size. Other selection methods may be used as well, forexample scoring, for example based on accumulated score of matching uppairs of triangles (this matching up may be thresholded duringconstructions, for example using a preset threshold).

Alternatively, other methods of finding a large common substructure areused.

It should be noted that while the clustering method may generate astructure that does not use all the triangles and is not complete, acomplete map of the pharmacophore is not essential for many embodimentsof the invention, for example for lead generation and finding.

7. Analysis

7.1 Overview

The above process of measuring and reconstructing a target area can beused to provide a wide range of information. The quality of theinformation and its type can be of varying kinds. Following areexemplary types of parameters which may be used to classify suchinformation:

(a) Completeness. The information may be complete or partial, forexample, a complete target area model or a model of only part of anarea.

(b) Factual or statistical. An example of factual information is anexact model. An example of statistical information is a set of relativeprobabilities for a set of possible models.

(c) Independence. Information may be independent of other information,for example, being an exact model or it may be dependent, for example aparameteric model whose exact value depends on additional information.In addition, information derived using the above methods may be used aspartial information for a different process.

(d) Substantiation. The information may be supported by otherinformation or it may stand on its own or even be in conflict with otherinformation.

(e) Positiveness. The information may be positive, in that it indicateswhat exists if is desirable, or negative, in that it can be usedprimarily to knock out certain possibilities.

While the information garnered may be about the binding locations, insome case, the information is regarding the geometry of the target atnon binding locations as well. As will be described below, for example,a geometrical structure can also affect the usefulness of a drug lead.

In some embodiments of the invention the analysis is used to acquireinformation about the gauges themselves, for example, their relativebinding affinity, and/or their chemical behavior (e.g., pHdependencies). Such information may be general or it may be for groupsof targets, for example, different for different families of proteinsand the same within a family.

As can be appreciated, such a widely varying range of information isamenable to many methods of analysis, some of which are described belowand to many applications, some of which are also described below. Inparticular, some exemplary analysis methods are directed to garneringfurther information about the target area and for error detection andanalysis and some exemplary applications are integrated as part of adrug discovery process.

In some case, the results of the analysis are integrated into thereconstruction as geometrical and/or chemical information. Alternativelyor additionally, the information is associated with the reconstructionand/or the target, for example, in a manner similar to that used fordrug leads. This manner generally depends on the type of database usedfor storing information.

7.2 Reconstruction Verification

In an exemplary embodiment of the invention, the error size and/or typeof the layout is determined. In one example, the reconstructed layout isanalyzed to generate theoretical binding values for the gauge set used.Differences between these theoretical binding values and actual bindingvalues may be used to indicate parts of the layout which are not exactand/or to indicate a degree of inaccuracy of the layout and/or thereconstruction process as a whole.

Alternatively or additionally, physical verification is applied, forexample, by applying an additional testing method and/or assay libraryto select between alternatives or for verification.

7.3 Binding Strength

In an exemplary embodiment of the invention, the generated layout isanalyzed to estimate the relative binding strength of binding points inthe target area. In an exemplary embodiment of the invention, thereconstructed layout is modeled and theoretical binding values for thegauge set are calculated. Variation in the actual binding values may bepartly caused by a reduced or increased affinity of target area. Suchestimation is generally statistical in nature since there are manyvariables that affect binding probability. However, it is expected thatif a bond length and type are known and the exact positioning of thegauge in the target area can be determined (e.g., and its energeticconsequences), than at least a statistical analysis of binding strengthmay be provided. Optionally, a baseline is provided by analyzingmolecules with known behaviors, or by comparing the binding ofdifferent, but similar gauge-triangles.

7.4 Inter-Bond Interactions

In an exemplary embodiment of the invention, the analysis is used todetermine an interaction between the binding of different bindingpoints. For example, such an analysis can compare the contribution of abinding point to the binding of a certain gauge, as compared to what isexpected (e.g., based on energy and other calculations) and/or ascompared to the apparent contribution of that binding point to thebinding of a different gauge. This may indicate, for example, the effectof the bonding to one interaction location on the affinity of aneighboring interaction location. Optionally such interactions areestimated and/or modeled using a model of electronic charge distributionin the target.

7.5 Geometric Analysis

For some purposes, and to some degree of accuracy, the determined layoutcan be considered to be a cast of the target area. In an exemplaryembodiment of the invention, the geometry of the target area isanalyzed. Additional information may be provided by determining whichgauges did not bind or bound with a lower affinity (which, if thebinding geometry was similar is assumed, in some embodiments of theinvention, to be due to steric clashes). This may assist in furtherdefining the geometry of the target area. It should be noted that somesteric clashes can be predicted from the geometry of the layout. Anyfailed binding which has no other apparent reason and should havematched the determined geometry, may be assumed to result from aprojection of matter that does not define a noticeable binding point.This is described in more detail below.

In an exemplary embodiment of the invention, the geometric analysis isused to determine a size of entry hole into area 104 (e.g., where arrow400 is shown in FIG. 4B). A small hole and/or certain moieties at thehole entrance may rule out the possibility of certain drug sizes and/ortypes. Alternatively or additionally, the geometrical analysis is usedfor classifying the target, for example, based on the size of substratethat it might work on. In an exemplary embodiment of the invention,geometrical analysis (e.g., for substrate determination) is supported bychemical analysis of the moieties in target area 104. Determination ofthe geometry may also be useful in deciding what marking methods ofsmall molecules and/or gauges may work (e.g., not to use largeflorescent markers, if the entry hole is small).

It should be appreciated that in some cases it may be easier toreconstruct the geometry of a target area, rather than its chemicalbinding pattern or vice versa.

7.6 Determination of Steric Clashes

In an exemplary embodiment of the invention, steric clashes are detectedin the analysis process and/or used to provide additional geometricand/or chemical information about the target. In an exemplary embodimentof the invention, steric clashes during the binding process aredetermined by comparing the affinities of different gauges with sametriangles. This comparison optionally takes into account one or more ofentry hole size, chemical behavior of the gauge, degree of matching tothe binding geometry and/or other binding locations. Steric clashes are,for example, caused when the proximity or potential overlap of the gaugeand the target molecule reduce the binding affinity.

As the shape of the gauges is known and, in some embodiments of theinvention relatively rigid, steric clashes may be expected to resultfrom the non-participating moieties of the gauge and/or the scaffolditself.

In an exemplary embodiment of the invention, the steric clashes are usedto generate a map of locations near the target that interfere with gaugeatoms, thus possibly indicating occupied (e.g., by atoms, electricfields) parts of the target, which do not, apparently cause a bindinginteraction with any gauge, to a noticeable degree.

In an exemplary embodiment of the invention, the map is used to providefierier information about the shape of the active area in target 100.Alternatively or additionally, the map is used for assisting in drugdevelopment, for example, by filtering out potential drugs that wouldhave the same steric clashes. Optionally, some level of filtering can beachieved simply by matching the drug geometry to the geometry of gaugesthat should have, but did not, bind well.

Geometrical and/or chemical affinity analysis may also be used todetermine a shape of the natural substrate of the target, for example,if it is not clearly known and/or to determine which part of thesubstrate is engaged by area 104.

7.7 Identification of Control Areas

In an exemplary embodiment of the invention, the binding results and/orreconstruction are analyzed to detect one or more control area of thetarget. Generally, control areas do not bind to the “main” substrate ofthe target, instead binding to a separate hormone or other modifiermolecule. This secondary binding typically affects the binding behaviorof the target area.

In an exemplary embodiment of the invention, control areas areidentified by their size and by their being disjoint from a main targetarea layout reconstruction. Alternatively or additionally, control areasare identified by testing bindings with pairs of gauges (or in thepresence of various molecules, optionally selected a-priori or after thedetection of the presence of control areas) to detect intra-gaugebinding dependence. Alternatively or additionally, control areas areidentified from the shape of the reconstructed layout. Alternatively oradditionally, the presence of control areas is detected by there beingleft-over gauge bindings that are not needed and/or do not fit in thereconstruction.

In an exemplary embodiment of the invention, depending on whetherbinding to a control area is desirable or undesirable, the differentialidentification of control areas may be used for screening potential drugleads.

7.8 Other Map Analysis

The map or model of the target may be analyzed to yield otherinformation, in accordance with exemplary embodiments of the invention.For example, as noted above, the distance of a binding point from acontrol area or active area can affect the type of drug developed. Forexample, a drug that binds in the control area may have an enhancingeffect on the target, for example that of an agonist. A molecule thatbinds near the control area or active area, or inside the active area,may cause the target to be less sensitive to signals and/or incapable ofacting, e.g., an antagonistic effect. Thus, in an exemplary embodimentof the invention, the location of the binding area on the target is usedto assist in determining what sort of therapeutic effect to expect froma developed drug. For example, a binding area near a target area mayindicate a drug whose tail blocks access to the target area.

In another example, binding areas that are outside the target area, canbe used to enhance a drug design. A drug may be constructed (ordiscovered) to include parts that bind in the target area and parts thatbind outside the target area. The combination of binding areas providesa binding strength greater than that provided individually by each area,while the part of the molecule bound in the target area can provide thedesired therapeutic effect. Alternatively or additionally, a moleculethat binds to two separate areas may cause a conformal change or preventsuch a change in the target molecule.

8. Use in Drug Discovery Processes

8.1 Overview

Drug discovery is a very long and expensive process whereby drugs forcuring diseases are found. The process starts with identifying a targetto be affected by the drug, finding potential drugs that affect thetarget and then determining which, if any of the potential drugs is safeand dependable. Often, no suitable drug is found and one of the drugcandidates is modified in various ways in an attempt to make it moresuitable. One cause of difficulty of the drug discovery process is thedifficulty in knowing what molecule will affect the target. As will bedescribed below, in some embodiments of the invention, the methods ofthe invention are used to at least partly reduce this difficulty.Another cause for difficulty is the many unexpected side effects ofpotential drugs which render them unsuitable and/or unpredictable.Again, as described below, some methods of the invention may be used toat least partly reduce this difficulty.

Typically, drug discovery methods try to answer two questions. One, isthere/what is a drug molecule that binds strongly and affects a targetmolecule. Two, how to ensure that these drug molecules have the properADMET profile (ADMET stands for Absorption Distribution MetabolismExcretion Toxicity) which translates into success in clinical trials. Inan exemplary embodiment of the invention, the method, materials and/orapparatus described herein are used to select, design and/or aim towardspotential drug molecules that have chemical characteristics that areknown or believed to improve the ADMET profile. Lipinski rules are anexample. It should be appreciated that by knowing which parts of themolecule are relevant to binding and which are not (as provided, forexample by comparing a molecule to a model of the target), one can moreeasily modify (or plan in advance) potential drug lead compounds to bindtightly and/or meet any well defined qualities.

In general, the above methods and especially the various models of thetarget can be useful in selecting molecules or research methods thatconform to the map and rejecting those that do not. Other uses of theabove measurement process are also described below, for example using anadditional step of mapping to check a theory. As can be expected,different methods (existing and new) of drug discovery may be affecteddifferently by the use of exemplary embodiments of the invention.

It should be appreciated that various embodiments of the invention maybe implemented in an automatic manner. However, due to the great costconsiderations, in some embodiments of the invention, the application issemi-automatic, for example, using the methods to change the discoveryprocess, for example, by adding a filtering step or a candidategeneration step, while still allowing for the use of human judgment, forexample, deciding if certain generalized and vague criteria are met. Insome case, the entire process is controlled using a human intelligence,with various ones of the steps, for example, mapping and/or rejectingleads are performed manually. Changing thresholds and redoing a step isan example of a decision which may be reserved for a human, for example.

8.2 Drug Generation

One relatively new type of drug discovery is actually drug generation, anew molecule is designed to have a desired function. In an exemplaryembodiment of the invention, the above chemical and/or geometrical mapof the target are used to assist this process. For example synthesis maybe assisted by showing what shape an active part of the drug must have(or limit the range of possible shapes).

In an exemplary embodiment of the invention, drug synthesis comprisestaking gauges from the library and modifying them, for example replacingmoieties, so that they better match the target. In some cases, thereplacement moieties have the same affinity but a different bindingstrength, for example, selecting NH2 or OH for a Hydrogen bond donor,and choosing an optimal size for a for hydrophobic moiety. It should beappreciated that an strength based classification of moieties may alsoused in the library construction, for example, providing multiplestrengths of Hydrogen donors or multiple sizes or hydrophobic moieties.One possible use is to achieve a better uniformity of binding strengthbetween moieties. Another is to provide a library with higher accuracy.

In an exemplary embodiment of the invention, scaffolds and/or moleculeparts for use in drug synthesis are constructed as a result of targetmapping processes. For example, by analyzing target geometries, a set ofscaffolds that spans (with attached moieties) most of the target spacesmay be found. The specific scaffolds may be, for example, constructedfrom sub-scaffolds or be selected from libraries of chemicals, forexample using a process similar to that described below for findinggauges in libraries. Sets of moieties or moiety clusters may beselected, for example based on a statistical analysis of how moietiesare clustered together in a family of targets or in a general list oftargets. Optionally, the statistics are collected over the mapping ofmany targets. Optionally, the targets are selected to be exemplary forexpected future targets. It is expected of course, that in some cases aperfect drug will not be generated using such synthesis methods, but thesynthesized drug may be a good starting point for drug enhancement.

8.3 Lead Generation

Often simpler than drug synthesis is lead generation, in which a lead,which is not expected to be a suitable drug, is generated and thenenhanced and modified using processes known in the art. In an exemplaryembodiment of the invention, the map is used to describe a potentialmolecule, for synthesis as a drug lead. In an exemplary embodiment ofthe invention, the map is used as a set of constraints and a search ismade to find a molecule meeting the constraints. Additional constraintscan be, for example, known synthesis methods, a base molecule form beingused as a starting point. An exemplary software which may be used isLUDI sold by MSI (USA). The LUDI system operates by attaching basicchemical components together in order to obtain a requiredpharmacophore-match or other molecule.

The potential molecule may then be synthesized and developed into adrug, as is well known in the art.

In an alternative method, a potential drug molecule may be constructedby linking together molecules of the gauge library or other moleculeshaving suitable moieties or structure, so that the resulting moleculehas a higher affinity than a single gauge. This molecule may then beoptimized, for example by removing unnecessary moieties and/or addingmoieties to provide various desired properties. Optionally, the gaugesare attached using a scaffold, rather than directly to each other.Optionally, by analyzing what gauges link (e.g., using clustering), itis possible to achieve a better estimate of a desired size and/orchemical properties of the fragments to be linked. For example, theselection of two gauges to be linked together may be based on actualbinding of additional (or other gauges), for example, 2, 4, 5, 6 or moregauges. For each such set of gauges that bind, a best gauge or othermolecule is selected for the linking. Alternatively or additionally,higher specificity gauges are used to determine which of the severalpossible triangle measures of a gauge actually bound. Such higherspecificity gauges may be generated, for example, by removing moietiesfrom existing gauges (or generating such gauges using any method knownin the art). Such higher specificity gauges may also be used for otherembodiments of the invention, for example, to improve clusteringstatistics. Generally, however, due to the relative large number ofpossible such gauges, they are used when there is a way to limit therange of possible triangles. Alternatively, the large number of morespecific gauges, for example, gauges with 1, 2, 3 or 4 triangles arecreated for use as a library or as part of a library of gauges.

In an exemplary embodiment of the invention, knowledge of the structureof the target is used to correctly locate the linker and/or chose asuitable linker that does not suffer steric clashes with the target.

In an exemplary embodiment of the invention, the gauges are selected forlinking without actually constructing a model. Instead, the actuallybinding gauges are selected and linked together. Alternatively, themodel is used to decide which gauges to link and how to link them. Sucha model may also be used in other lead-linking schemes, for example, asdescribed in the background, to guide the choice of which fragments tolink, what length of link to provide, where to attach and/or at whatorientation to attach. Optionally, the lead is constructed in steps fromthe gauges, and each step is tested to see if it meets its expectedbehavior.

Alternatively or additionally, instead of using a model as providedherein, a different type of model is used, for example a model of thetarget bound to a gauge, for example generated using X-rayCrystallography and/or NMR. This model, for example, generated once foreach of a plurality of gauges may be used to decide what linkingdistance and type to provide when creating a lead from gauges.Alternatively or additionally, a new molecule may be designed andconstructed to have binding points at some or all of the locations shownby the crystallography model to bind, for example, for two, three ormore gauges. In general, this type of method assumes that once theconfiguration of the bound target-gauge pair is known, an exact modelmay be unnecessary, since actual conformance information is available.Alternatively or additional, as noted herein, a measurement may be madeof the bond target.

8.4 Lead Description

In an exemplary embodiment of the invention, the map is used to describeone or more profiles of molecules which would be expected to have aneffect on the target. In an exemplary embodiment of the invention, theprofiles generated take into account one or more of:

-   -   (a) geometry of interaction location layout;    -   (b) affinity of interaction locations;    -   (c) size of entrance into the active area(s);    -   (d) identification of potential control area(s);    -   (e) synthesizability; and    -   (f) expandability, e.g., that additional moieties can be        attached.

Typically, a molecule requires at least five or six bonds to form astrong enough docking in the target, so as to affect the a target atnano-Molar concentrations. The exact number may depend, for example, onthe affinity of the interaction locations. A single target willgenerally provide a large number of possible profiles. These profilesmay be matched against libraries, for example, using methods known inthe art.

In an exemplary embodiment of the invention, the profiles are generatedusing a format that is matched for a particular search software and/orlibrary data structure. In an exemplary embodiment of the invention,searching by pharmacophore is provided, for example as known in ISISbase from MDL (when searching 3D databases).

8.5 Lead Search

In an exemplary embodiment of the invention, the map is used to searchthrough a library of known molecules, for a possible match. Possibly,the map is used in place of analytical models of the target, in knownvirtual scanning techniques. In an exemplary embodiment of theinvention, the library is pre-processed so that molecules in the libraryare described in terms of the moieties and geometries of the layoutmodel and/or the gauges used in measuring the target. Alternatively oradditionally, an existing library is pre-processed to yield agauge-compatible description of its contents, for example, each moleculebeing defined as a parametric model based on measurement gauges. Itshould be noted that this description may not be a one-to-one mapping,for example, a same molecule may be described using two different setsof moieties, as there is some overlap between moieties chemicalbehavior.

In an exemplary embodiment of the invention, potential leads areidentified based on them including or being able to include a largenumber of moieties at the required positions, as indicated by the map.In one example, a search is made for 3 point or higher (e.g., 4, 5, 6, 7or more) matches. In another example, each molecule in a library istested for the number of moieties it includes in the required positionsand for the availability of attachment points to attach missingmoieties. In an exemplary embodiment of the invention, the missingmoieties are added one by one until a suitable drug lead (e.g., strongenough binding) is created.

An exemplary search is performed by ISIS base, by MDL.

One possible type of search comprises going over all available 3Dstructures in which to search, breaking them down into sets and/orsubsets of pharmacophore points and looking for a fit within a tolerancerange defined in a query.

8.6 Lead Rejection

In an exemplary embodiment of the invention, the results of the abovemethods are used in rejecting leads that otherwise appear suitable. Inone example, a lead (or family of leads) is rejected if the above modelsimply a lack of binding and/or steric clashes. En another example, anassumption is made that if a lead is suitable, a gauge corresponding toa triangle (or other number) of moieties on the lead is expected to bindto the target. If no such gauge is found or an analysis of the dataimplies that the probability of a triangular binding of the threemoieties in a gauge is unlikely to have happened, the lead is rejected,or subjected to additional scrutiny. Alternatively or additionally, amatching of a certain gauge may also indicate the lead is unsuitable.

In one example, workers in the field can use the information provided todetermine if a certain lead is likely to be capable of being optimized(reasonably). For example, one expects that by directly adding orremoving specific moieties (e.g., what is often considered to be a maintype of small changes in a molecule) affinity can be significantlyimproved (often at least 3-4 orders of magnitude are required). Byknowing what the changes should be or could be (e.g., where additionalpoints need to be added, which information may be provided by someembodiments of the invention) one can see if one specific lead canundergo the required changes, e.g. has putative attachment points in theright positions. Specific gauges (e.g., that bound) will indicate whatthe required changes could be.

These methods may also be used to reject certain lead modificationsprovided during lead enhancement processes.

It should be noted that some lead rejection methods do not require allpossible gauges and/or triangle measures to be available. Rather, even apartial library is useful, for example for rejecting certain leads. Inone example, a partially-spanning library is used generating a partialmap (e.g., part of space, disjoint and/or not all binding points), whichcan be used to reject some leads and/or indicate potential suitabilityof others and for search. Further, even the binding or failure ofbinding of a single gauge may indicate suitability or lack ofsuitability of a lead. Generally, due to the uncertainty involved in allchemical processes at this time, decisions are not made on the basis ofa single binding assay.

8.7 Targeted Mapping

In some embodiments of the invention, gauge binding is assayed duringthe discovery process. In one example, the binding is used to test atheory or an assumption regarding the leads. For example, if a certainlead is expected to be suitable, at least one of several particulargauges may be expected to bind. Leads may be ranked, for example, basedon how well such targeted binding is. Alternatively or additionally, apart of the layout may be remapped as a result of the discovery process.For example, the discovery process may indicate conflicting evidence ofthe layout structure. In another example, a higher resolution mapping ofpart of the layout may be required, for example, to more exactlydetermine the distance between two moieties. In some cases, instead ofassaying with a full set of gauges, gauges are selected based on thembeing most likely to bind (or not) to the particular desired parts ofthe layout. For example, if the distance between two points on thelayout needs to be determined, gauges that are less likely to bind atother points of the layout are selected. In another example, themoieties used are more specific, for example, having a more limitedrepertoire of chemical behavior and/or have a greater directionality.This may require using a different scaffold. Possibly the gauges usedfor such remapping have fewer triangles per gauges, for example betweenone and three, to reduce unexpected binding probability. Alternativelyor additionally, gauges are selected so that steric clashes preventbinding in undesired locations. In some cases, these gauges are not inthe basic mapping library used for initially determining the layout. Insome cases, the required gauges are synthesized ad hoc, rather thanselected from an existing library.

8.8 Target Suitability Testing

In an exemplary embodiment of the invention, the map is used todetermine the suitability of a target to be a target for a drug. Asuitability value may be, for example binary or it may be graded(discrete or continuous). In some embodiments of the invention, asuitability value is not a scalar, for example, being a vector, witheach element of the vector indicating a different aspect of suitability.A similar structure may be used to indicate suitability of leads andpotential drugs.

One example of the use of target suitability testing is where there aremultiple potential targets. For example, in some diseases, there is apossibility of selecting between a plurality of target proteins, orselecting different parts in the chain of protein synthesis (e.g., DNAtranscription, protein-chain creation, protein folding, proteinpost-processing and protein deployment). Some of these potential targetsmay be unsuitable.

In an exemplary embodiment of the invention, the map can be analyzed todetect such suitability, for example, by rejecting targets with anactive area that is too large (for some types of treatment). The size ofthe target area can be detected from the layout geometry. Alternativelyor additionally, a target may be deemed unsuitable due to its having atoo generally active (non-specific) target area, which can bedetermined, for example, by analyzing the specificity of the determinedtarget layout. Alternatively or additionally, a target may be deemedunsuitable, because its active area that has very weak affinities (e.g.,a large drug molecule with many binding points may be required).Alternatively or additionally, a target may be deemed unsuitable due toits similarity to a housekeeping protein. This similarity may bedetermined by comparing the layouts of the target with those of knownhousekeeping proteins. Similarity to any human protein may assist indetermining potential side effects ahead of time. In lead grading, alead may be evaluated based on a probability of its interfering with ahousekeeping protein, which is optionally determined by checking thebinding of a lead to model layouts of housekeeping proteins.

In an exemplary embodiment of the invention, a database of layouts ofhousekeeping proteins is provided, such a database may be provided usingmethods known in the art. Alternatively or additionally, at least partof the database is provided by systematically mapping housekeepingproteins. Alternatively or additionally, at least part of the databaseis provided by generating “worst case” target area geometries or a rangeof possible geometries for the active areas, based a knowledge of thestructure of substrates that are acted on by the protein. Such a worstcase target area may also be used as prior information to assist indeciding which of several reconstructions is correct.

8.9 Target Partitioning

In an exemplary embodiment of the invention, the map is used to identifyparts of the target as being potential “exact” targets, and on which thedrug discovery method can be focused. Although the target, as a whole,is what is being affected by the drug, it can be affected in many ways,for example, different drugs may block different parts of an activearea. Alternatively or additionally, some drugs may cause conformalchanges. Alternatively or additionally, some drugs may interact withcontrol areas on the target. Alternatively or additionally, some drugsmay be agonistic, while some antagonistic. Alternatively oradditionally, some binding areas may be useful for staging (e.g., as abase for attaching molecules closer to a target area), rather thandirect activity. Binding areas may be classified based on the type ofeffect that may be expected from a molecule binding to those areas. Thisclassification may be, for example, manual. Alternatively oradditionally, automatic classification may be provided, for example;based on target template structures (e.g., which indicated for a certainclass of protein what each area of the protein might do).

Optionally, potential control areas that can change the target, areidentified. Possibly, such control areas are identified based on abinding in a binding assay. Optionally, a model of the target is used toassess whether binding at the potential control location can causeconformal changes, for example, based on the proximity of interactionlocations on different, adjacent parts of the protein.

In an exemplary embodiment of the invention, the active areas aresegmented into different “exact targets” based on the sub-areas thathave a potential for drug interaction, for example, based on theirgeometry. Alternatively or additionally, segmentation is based onselecting such sub-areas that are not common to similar sub-areas ofhousekeeping proteins (e.g., segmentation into special and commonbinding areas).

8.10 Drug and Lead Analysis and Enhancement

In an exemplary embodiment of the invention, the above layout is usedfor analyzing existing drugs or drug leads, for example, to assist inimproving or reengineering the drug or in screening.

In an exemplary embodiment of the invention, the layout is used todetermine which of a plurality of target areas on a target areinteracting with the drug or which target from a plurality of possibletargets are interacting with a given drug. This methodology may be used,for example, to analyze the effect of drugs whose operation method isnot clear.

In another example, the drug is analyzed to determine which part of thedrug binds to the target. This can serve as a basis of a process formodifying the drug, in which the binding parts of a drug are retainedand other parts of the drug are modified. Alternatively or additionally,when modifying the drug, care is taken not to distort the active part ofthe drug so that it does not bind, or distort the drug as a whole sothat steric clashes are caused.

It should be noted that a single drug may interact with two differenttargets in a desirable manner, each target interacting with different,possibly overlapping, parts of the drug. Such activity of a drug isoptionally determined by comparing the drug structure to that of thetargets.

In some cases, the exact spatial and chemical properties of the drug (ora protein substrate) are not known. However, by determining the layoutof targets which bind to the drug, the spatial and chemical layout ofthe active part of the drug (or substrate) may be estimated.

In another example, the layout is used to determine the pharmaceuticalactivity of synthesis byproducts. When a drug is produced using aparticular process, various byproducts are produced as well, some with abeneficial activity and some with a non-beneficial activity. In anexemplary embodiment of the invention, the structure of such byproductsis compared to target areas of the target and of housekeeping proteinsin an attempt to estimate what side effects they might cause. A processfor drug manufacture is optionally selected or rejected based on athus-estimated activity of the byproducts, given that the type andamount of byproducts produced by a particular process can be determined.Alternatively or additionally, such comparison may be used to assist inimproving a production method and/or in deciding which synthesisparameters to use. This testing may also be used for regulatorypurposes, for example to approve or disapprove generic drugs.

8.11 Drug Selection

In many cases, there may be multiple drugs which can treat an illness.Knowledge of which target (and housekeeping proteins and/or other humanproteins) is affected by a drug and how it interacts can be useful inselecting between alternative treatments, in preventing side effects,preventing or controlling drug-interactions and/or in selectingtreatments for diseases that no exact drug has been selected for, forexample exotic tropical diseases and some viral diseases.

In an exemplary embodiment of the invention, the layout of a target isused to select which of a plurality of available drugs or drug leadsappears to be most suitable for interacting with a the target. In thecase of drugs, this may allow selecting alternative treatment protocols.Also, in some cases, knowledge of the interaction method will assist inselecting those times and/or associated protocols and/or drugcombinations at which the drug is most effective and/or has minimal sideeffects.

Alternatively or additionally, drugs may be designed to interact withmultiple targets. For example, a lead that interacts with multipletargets (e.g., of a same or different disease or syndrome) or targetarea portions may be awarded a higher scoring for further processingthat other leads.

A possibly related use is the finding of a new use for an old drugand/or assisting in determining how to modify an old drug for a new use.For example, when searching for leads that match a template, a searchmay also be made through databases of drugs, to see which drug has astructure that is prophesied by the modeling process to provide goodbinding. Existing drugs, in general, have the other properties (ADMET).

8.12 Drug Enhancement

As noted above, knowledge of the interaction method and/or problems ofinteraction with a target area, can assist in modifying a lead to becomea drug. Alternatively or additionally, such knowledge may be put to usein enhancing an existing drug and/or modifying a drug to interact with atarget related to an existing target. By comparing the layouts of thetwo targets, for example, possibly useful changes in a drug may bedetermined. Alternatively or additionally, the layout of the target areamay be used to assess problems with the binding of the drug to thetarget (e.g., too strongly or too weakly) and/or determine the effect ofmodification of the drug on such binding behavior. In an exemplaryembodiment of the invention, the potential drug when bound is assessedagainst the model, to determine if a moiety exists that can betheoretically added, which will bind to another point in a binding area.

Alternatively or additionally, drug enhancement comprises enhancing adrug to match more than one target, or a variety of target mutations,for example including one moiety to bind for one mutation and one moietyto bind for another mutation, for example, in HIV some proteins have twomain varieties and countless sub-varieties. This enhancement mayinterfere with other properties of the drug, but the tradeoff may beconsidered useful.

Alternatively or additionally, a drug may be designed to bind to asubset of binding points that is common to a plurality of targets ormutations, for example, models of the plurality of targets are analyzedto determine shared binding points. The various drug discovery methodsare then optionally applied assuming that only these binding pointsexist. Real assaying of a potential drug may be carried out on themultiple targets to ensure that the various modifications of the drugdid not make it fail to bind to one of the targets. Alternatively oradditionally, when a modification is made, it is determined whether themodified drug will bind to the common binding locations and/or havesteric clashes. It should be noted that there might be other reasons todiscover a drug that binds only to a subset of the possible bindingpoints, for example, if a mutation is expected in one of the bindingpoints and/or to allow the drug to work even if an interfering moleculeis bound to one of the binding points.

8.13 Drug Failure Analysis and Reengineering

Often, a drug will come out to market and then fail. The methoddescribed herein may be useful in determining a reason for the failureand then possibly assisting in rescuing the drug. In an exemplaryembodiment of the invention, the layout of the target of the drug and/orother proteins that the drug is believed to have interacted with (e.g.,based on the type of side effects), are generated. The drug is thencompared to the targets to determine failures in binding to the correcttarget and/or undesirable binding to non-targets. It should beappreciated that while such comparison may be theoretically possibleusing other means, it is believed that prior to the availability oftarget mapping, such large scale molding of active areas of targets wasnot practical, due to time and cost limitations.

In an alternative embodiment of the invention, it is noted that a drugmay be suitable for only part of the public, for example, due toindividual differences. In an exemplary embodiment of the invention, thegenes that express inadvertent targets and/or targets are used toreconstruct models or samples of the targets and then map the activeareas of the models. The results may show that an individual has asensitivity to the drug and/or that a different individual is resistantto the effects of the drug. Alternatively or additionally, the testingmay be done against pathogen strains, to determine differentialsensitivity to drugs. In some cases, the genetic differences are linkedto already known markers, for example sensitivity to sulfates is linkedto a G6PD deficiency for sulfates, so that the classification of peopleas being compatible with the drug may be simple. Alternatively, agenetic test may be applied prior to selecting which drug to use on aperson.

8.14 Additional Drug Discovery Related Analysis

Additional analysis methods may also enhance a drug discovery process.For example, many drugs have side effects due to their interaction withhousekeeping proteins or proteins that cause feeling of malaise ifinterfered with. Examples include GI proteins and liver proteins. Somedrug targets are known to be similar to such proteins. In an exemplaryembodiment of the invention, models are generated for such potentialside-effect generators. Any potential drug lead is rejected (or scoreslower) if it is shown to bind to one of these prohibited models.Alternatively or additionally, drugs that have a known side effect areanalyzed to determine which protein they bind to and this protein and/orthe particular binding locations are used for defining a prohibition ofbinding of a potential drug.

In another example of an analysis, potential drug molecules are analyzedto see if they bind as a substrate to certain enzymes. Such binding mayindicate a speed of incapacitation of a drug or its excretion.Alternatively or additionally, such binding may be useful foridentifying pro-drugs, that are activated by their interaction withcertain enzymes, such as liver enzymes. In this case, a drug may includetwo sets of active areas, one for activation of the drug and one forbinding of the drug to its target. Optionally, biding to a protease (orother manipulating protein) is ensured by adding binding moieties orgauges to a drug molecule at suitable locations.

In another example, a set of target molecules that are all known to beaffected by a same protein or molecule are analyzed to determine of theyhave common binding geometries to which the molecule bonds. This mayhelp, for example, in fine tuning the molecule to bind more selectively,for example, by adding a moiety which will interfere with other targetmolecules and/or assist in binding to a particular target molecule.

8.15 Streamline Discovery Process

As can be appreciated a discovery process typically includes goingthrough various dead ends. In an exemplary embodiment of the invention,mapping of the targets is used to select parts of the discovery processthat are likely to fail and prevent them from being attempted. Someexamples (some of which are described elsewhere in this application)include, dropping targets that do not seem suitable for improvement,identifying targets likely to have side effects and weeding outlibraries. In an exemplary embodiment of the invention, weeding outexisting libraries is performed by removing from a library leads thathave an expected low probability of binding and/or appear redundant toother molecules. For example, a molecule that is very flexible is lesslikely to bind. The probability of binding may be estimated, for exampleusing energetic considerations based on the molecule's degrees offreedom.

8.16 Utility Generation

While many proteins and molecules are catalogued, many of them do nothave a known utility. Determining an exact utility for a protein or amolecule may require a very large expenditure. In an exemplaryembodiment of the invention, potential utilities for molecules and forproteins may be generated on a large scale in the following manner. Amolecule may have a utility as a gauge or it may have a utility as alead or drug. La an exemplary embodiment of the invention, existingtarget area layouts, for example, 10, 50, 100, 1000 or any smaller,greater or intermediate number are matched to the molecule to see ifbinding is likely. It is expected that many molecules will turn out tohave a potential utility. In general, more matching is more work, butincreases probability of success.

In a similar manner, mapping proteins provides an indication of itsactive area shape, potential substrates and/or potential drugs whichmight affect it. In an exemplary embodiment of the invention, a utilityis found for a protein by determining its substrate. Optionally, theprotein active area layout is compared to structures of known substratesand proteins.

In this manner, a library and individual drugs and proteins may be saidto have an expected utility. For example, the protein may be for one ofthe following protein families GPCR's, Proteases, Kinases, Ion Channelsmessenger proteins or any type of peptide or other macro-molecule foundin a living organism.

9. Exemplary Discovery Applications

9.1 Overview

In this section existing discovery methods will be described, as well aspossible modifications that take into account the methods describedherein.

While many approaches to drug discovery are known, the following twomain approaches generally encompass the existing methods.

9.2 Screening Based Drug Design

This discovery method works by screening a target against a large numberof molecules and then attempting to enhance any matches to produce adrug. The process is as follows:

(a) Provide a general library of compounds for screening, equallyrelevant to all target proteins. Typical sizes of such libraries growconstantly at roughly one order-of-magnitude (factor of 10) per decade.Current typical sizes are 1-10 million. The libraries are oftenproprietary and maintained by each corporation independently.

(b) Screen the corporate library against the chosen target. Look forcompounds exhibiting at least weak activity (significant activity atconcentrations typically 1-100 μM) of the type required with relation tothe target.

(c) If no hit is found, the process ends here. Apparently, this is oftenthe case, possibly in above 70% of the cases. If hits are found, anoptimization stage is initiated, in which the final outcome is expectedto be a compound with strong activity (at concentrations typically n)against the target. This is done in one or a combination of thefollowing two methods:

1. In case there is only one hit or all hits are variations of onemolecular theme, a large number of analogues of the hit are synthesized.This group of compounds is sometimes known as a “focused library”. Theseare also screened against the target protein. The purpose here is todefine a direction for increasing the activity of the original hit byidentifying chemical moieties and positions on the original hit thatincrease activity. This process is known as developing a QSAR(Quantitative Structure Activity Relationship).

2. If a number of chemical groups have been identified as hits, acomputational process of identifying possible pharmacophores (molecularsubstructures directly involved in binding of the hits to the target) isexecuted. These may indicate not only possible directions foroptimization, but also their feasibility for a given molecular startingpoint (both from a physical point of view and from a synthesis point ofview).

(d). Drug like qualities are generally a byproduct of this process.Molecules in the initial screening library are often chosen to possessdrug-like qualities. During the optimization process, only partialinformation is available so that simultaneously satisfying drug-likerequirements and increased activity are seldom under direct control.Final drug-candidates that may result from this process closely resemblehit compounds in the initial screening library.

(e) Testing. The drug-candidates are tested, for example in live animalmodels and then in humans, to determine there efficacy. Many drugcandidates' fail at this point and lacking any basis for modification,fail completely.

In an exemplary embodiment of the invention, the above describedinventive methods may be used to improve the above drug discoveryprocess, for example one or more of:

(a) Hit rate. As stated above, in most cases, no hits are found for anew target. By generating a mapping of the target, the leads used forscreening can be better selected. Even leads with very weak affinity maybe selected for further improvement, due to the combined indication ofvery weak activity and matching a map. Alternatively or additionally,the method of designing a gauge library is applied to a moleculelibrary, to reduce duplication and to assist in ensuring coverage ofbinding space. This may be done, for example, by analyzing the libraryto identify gauges in triangle space and/or uneven distribution leads inthis space. In addition, excessive overlapping may be determined.Alternatively or additionally, the library may be analyzed to determinemolecules that are unlikely to ever bind, for example, due to themhaving excess flexibility and no known binding partners. Alternativelyor additionally, if the screening is in stages, molecules may beselected for each stage based on them having less overlap with eachother.

Alternatively or additionally, some binding results may be ignored, forexample, molecule with high flexibility may add too much noise (bindingto many molecules in many ways) and therefore be ignored, at least in afirst stage of processing.

Alternatively or additionally, the gauges that bind can themselves beused as leads (and many of such bindings are expected). Often the gaugelibrary is small compared to the corporate library and can be added toit with a relatively small penalty. In an exemplary embodiment of theinvention, results from the “old” library will serve as initial startingpoints for optimization (as before) but optimization will be directed byinformation gained from screening using gauges. Possibly, a gaugelibrary binding assay is performed on a target with an interacting lead.This type of assay can be used to determine if the lead (or moleculefrom a library) is interacting with the active area or not (e.g., basedon whether and the extent that it affects the binding of the gaugelibrary). This assay may be compared to an assay performed with otherbinding leads and/or with no bound leads at all. The effect of leadchemistry may be determined by checking the assay in the presence of oneor more chemically similar but non-interacting leads.

(b) Process directing. If the target is mapped and a lead starting pointis known, there are still many ways of enhancing the lead to produce adrug. In an exemplary embodiment of the invention, knowledge of thetarget geometry and/or chemical behavior is used to assist in directingthe modification process, replacing physical experiments with virtualones and/or assisting in culling out (probably) useless leads. Inaddition, it is noted that various combinatorial generation of leadmodifications can be simplified by selecting only those leadmodifications that are meaningful (or are most meaningful) in view ofthe target layout and/or based on the three-dimensional structure of theleads (e.g., by checking which triangles are exhibited by which lead andby which lead modification). Optionally, a mismatch between the resultspredicted by the determined layout and actual binding activity of theleads may be useful in correcting the layout, better understanding thechemistry of the lead and/or predicting other leads that might showpromise.

(c) Drug recovery. Even if a drug fails the final testing stages, in anexemplary embodiment of the invention, the above methods may be used todetermine the reasons for the failure and/or provide guidance inreengineering the drug.

9.3 Alternative Screening Based Drug Design

Chemical genomics or chemogenomics have lately become very popular. Theyare based on the idea that instead of first finding a target first andthen finding a compound for it, the opposite process is applied: firstscreen compounds against whole cell assays looking for the phenotypicresult (e.g., selective death of cancer cells). Then, once an activecompound is found, the target is sought. One possible advantage of thisapproach is working in parallel on multiple targets, many of which maynot even be known. However, existing screening libraries cannotguarantee finding hits. In an exemplary embodiment of the invention, agauge library as described herein is used and is expected to have aplurality of gauges that interact with the cells. While the interactionsmay be weak, a non-trivial number of such interactions may be expected.

9.4 Structure-Based Drug Design

This method assumes that accurate modeling software for simulatingmolecular processes is used. The process is as follows:

(a) Obtain an accurate and detailed three-dimensional structure of thetarget protein. Usually done via X-ray crystallography or NMR analysis(both experimental). Computational approaches also exist, but aregenerally not accurate.

(b) Identify the active site in the protein structure (not alwaysstraightforward for new, unfamiliar targets).

(c) Identify relevant binding points in the active site, also known aspharmacophore points. These are points where weak (non-covalent) bindingcan occur. A potential Ligand must satisfy a number (usually 6 or more)of these points simultaneously in order to achieve nM affinity.

(d) Design molecules that “fit” the active site, both geometrically andin terms of satisfying enough pharmacophore points. Both this stage andthe previous are done using “docking” or molecular-mechanics typesimulation software.

In an exemplary embodiment of the invention, the herein describedinventive methods may be used to improve the above drug discoveryprocess, for example one or more of:

(a) Linked structure. 3D structures of proteins are apparently, in manycases, of little use in and of themselves. Much experience has shownthat it is difficult to design strong binders based on this (e.g.,geometrical) information alone. In an exemplary embodiment of theinvention, it is noted that useful information is present in 3Dstructures of the target with bound ligands. While such ligands are notknown initially, in an exemplary embodiment of the invention, gaugesthat bind to the target are used in place of such ligands, with theexpectation that a significant number of such binding gauges will befound. In an exemplary embodiment of the invention, the gauge bindingprocess is applied and then the target is modeled (e.g., using NMR orX-ray crystallography), possibly several times, with different gaugeslinked. The shape of the target area with the linked gauges is expectedto be useful for designing strong binders using methods known in theart. Possibly, the known methods may be modified, for example, tocombine the results of different configurations caused by differentbinding locations of different gauges. Optionally, the provision ofmultiple binding gauges (e.g., 5, 10, 25, 50, 100 or any smaller,intermediate or larger number) will assist in determining the bindingmode(s) of the target, possibly enhancing the understanding by providingpartial binding modes as well. In general, the provision of more gauges,means more work, but may enhance the accuracy of the analysis.

In an exemplary embodiment of the invention, the linked structureresults from a plurality of gauges are combined, for example by superposition with the target as a reference. This superposition may yield atotal model of the binding area of a target and/or fully boundconfiguration, rather than a partial one might be provided by eachgauge.

(b) Comparison. In an exemplary embodiment of the invention, the shapeof the active area determined by the simulation model is compared to theshape of the area as determined by the mapping process. Differencesbetween the two may assist in correcting the mapping/reconstructionmethod or it correcting the simulation model. Optionally, the simulationmodel is used to select between alternative reconstruction and/or toassist in fine-tuning a reconstruction, for example, by assisting incalculating more exact distances and/or indicating which possiblemoieties could be taking part in the binding.

(c) Identification of binding points. In general, modeling software isnot accurate enough to predict binding points in a protein target. Alsoactive areas may be difficult to identify. This is especially the casefor novel targets. In an exemplary embodiment of the invention, theabove methods circumvent one or both of these problems by identifyingpotential binding points/modes experimentally, e.g., using a standardassay library of gauges. Then these active areas are analyzed in greaterdepth using docking software, for example to predict the affinity of newcompounds to a specific target.

9.5 Modular Assembly of Ligands

This method, which is apparently used by Sunesis inc., works byconstructing leads from parts that show affinity. The process is asfollows:

(a) Synthesize a finite library of elementary molecular fragments thatinclude a “linker port” (i.e. a site on the molecule at which linkingcan be easily implemented). These are typically small moleculespreviously identified as pharmacologically “interesting”, and which areamenable to including the standard “linker port”.

(b) Screen the elementary fragments against the target protein, lookingfor extremely (˜1 mM) low affinity. This step is typically problematic.

(c) Link groups of two or more fragments via their “linker port”components in order to achieve increased affinity. The distance betweentwo fragments, i.e. the length of the linking chain, may be varied andoptimized.

In an exemplary embodiment of the invention, the herein describedinventive methods may be used to improve the above drug discoveryprocess, for example one or more of:

(a) The elementary fragments are currently not designed in the art usingany logic that may be viewed as exhaustive, i.e. typical diversitymetrics are used (as in standard screening libraries) but these do notyield a finite list. Consequently, hits are seldom found (for generaltargets), even less than for general screening libraries, probably dueto very low affinity expected, which poses many technical problems (e.g.solubility). In an exemplary embodiment of the invention, the set offragments is selected based on spanning the space. For example,fragments may be pairs (or triplets) of moieties, having distances andmoiety types selected to span the possibility space.

(b) Geometry, i.e. the proper distance and orientation between twoweakly binding moieties, is totally absent from the initial screeningresults in the art. In the linking stage, only very limited geometryvariation may be tried (i.e. the length of the linker). In an exemplaryembodiment of the invention, the binding of a gauge library is used toprovide geometrical hints (or a complete model) which assist in decidinghow to put together fragments, which fragments to put together and whatdistances to set between the fragments. This may also assist indetermining what type of linker to use when linking fragments. This mayalso be used for synthesizing a new molecule that includes the bindingparts of the binding gauges, spaced apart by a suitable structure (e.g.,a variation on a known drug).

10. Exemplary Non-Discovery Uses

The above measurement methods may also be applied to uses other thandrug discovery. A different gauge set may be required for some uses.

In one exemplary embodiment of the invention, the measurement methodsare used to assess toxicity, for example, to identify housekeepingproteins that may have adverse interactions with a certain drug orpotential toxin. This may be useful in determining toxicity ofindustrial or household chemicals.

In another exemplary embodiment of the invention, the measurementmethods are used to predict antibody affinity to a material and/or cell,for example by identifying binding sites on an antibody and/or amaterial.

In another exemplary embodiment of the invention, the measurementmethods are used to map the outside of an organism, for example, avirus, rickettsia bodies, worm, protozoa, fungus, ameba or a bacteria.This may be useful in the development of vaccines. For example, avaccine is often more effective if it is made from a protein whose shapedoes not change. By determining which parts of the binding areas on theoutside of a pathogen do not change, such determination may assist inselecting a particular protein from the pathogen for vaccination useand/or to assist in assessing the chances of creating a useful vaccine.In order to prevent auto-immune responses, the active areas of existingvaccine material may be mapped, to see if the pattern resembles that ofbodily proteins to too great an extent. It should be noted that thismatching may be dependent on an individual's genetic material.

Alternatively to absolute measurements, in some embodiments of theinvention, the above methods are used for determining relativemeasurements, for example, for measuring conformal changes in a protein,under different conditions. A same (or different—e.g., to match newexpected measurements) binding assay may be applied to the protein underdifferent conditions. Possibly, more flexible gauges and/or less stablegauges are used for this application.

In another exemplary embodiment of the invention, the above measurementmethod is used to find new agricultural chemicals, such as insecticidesand herbicides that are target-specific by affecting proteins known tobe crucial only for some types of pests or weeds. Alternatively oradditionally, artificial hormones are developed to match targets inplant cells.

11. Using Prior Information

The above process has been described, in some examples, as a blindprocess, which assumes a neutral starting point of substantially noknowledge about the target. In some cases, there exists prior knowledgeabout the target, gleaned from various sources and/or by previousmeasurements of the target. Such prior information may be used in manyways. Following are some examples.

In an exemplary embodiment of the invention, the prior information issufficient to propose several alternatives. A binding assay with thegauge library, with or without reconstruction may provide enoughinformation for selecting between the alternatives, for example betweenalternative models of which part of a lead interacts with a target orselecting between two target area layout reconstructions. Optionally, tothis end, the gauge set can be reduced to only those gauges that willdistinguish and/or that are needed by either one of the models.

In another example, crystallography, NMR, IR spectrum and/or chemicalproperties of the target are used in the above reconstruction process,for example, to resolve ambiguities and/or to overcome lack of data. Inone example, these methods show how one or more gauges actually bind inthe target. In another example, these methods or other prior knowledgeare used to force a certain structure to be reconstructed, rather thanfollowing the above described score based reconstruction. For example,forcing the structure to include a certain sub-shape (e.g., atetrahedral portion) that would not otherwise be reconstructed from theassay data.

In another example, if part of the target is known, it can be reactedwith a substrate that blocks out that known part, so that themeasurement will only apply to the unknown portion. Alternatively, thestatistics of interaction in the known portion may be used to assist inassociating binding statistics with structure in the unknown portion.For example, a computer model or an analogue target may be used toprovide an estimate of which gauges bind and at what strength, to theknown portion. In the assay results analysis, gauges that bind to theknown area are ignored, not used in the assay and/or their bindingstrength reduced during the analysis. Optionally, a gauge is not removedfrom consideration if removing it will leave no triangles of a certainsize and/or moieties for binding to the unknown area. Alternatively, thelibrary as a whole is used, for example, as noted above thatsimultaneous screening using 100,000 assays at a time, is a currenttechnology.

In another example, when an iterative measurement method is used, priorinformation may provide insight into desirable starting points.

Optionally, the prior information is used as an input for modifying thebinding process, for example by varying the binding environment.

In another example, the prior information is used to set theenvironmental conditions used during measurements, for example, usinginformation from previous assay attempts with a similar protein toindicate what environmental conditions are likely to provide bindingsand/or at least not interfere.

In an exemplary embodiment of the invention, prior information is usedfor the design of specific scaffolds, moieties and/or gauges to bettermeasure a particular target. The molecules may be, for example, designedad hoc, and/or a sub-library constructed by selecting previously knownmolecules. In an exemplary embodiment of the invention, a scaffold isselected for such a sub-library due to a small (e.g., 0.5 Å) differencein a side of a triangle due to the change in scaffold. In a regularmapping process, such a difference may not be important, but inhigh-resolution mapping, for some targets (e.g., where binding is weak)it may be important. Similarly, a set of gauges may be provided to covera certain range of sizes and/or chemical behaviors at a finerresolution.

12. Iterative Measurement

In some ways similar to the use of prior information, iterativemeasurement allows information form a previous measurement step to beused, for example, to better tune a current step or to reject certainpossibilities.

In some embodiments of the invention, instead of a one step measurementprocess, for example as described in some of the embodiments above, aniterative measurement method is used. In one example of this method, alower resolution reconstruction is generated. Then additional assayingis performed, using a same or different gauge library and a higherresolution reconstruction is provided. The earlier reconstruction may beused, for example, as a starting point for the reconstruction processand/or to assist in selecting which gauges to use in the additionalassaying. In an exemplary embodiment of the invention, such an iterativemethod is used, for example, when the cost and/or time to perform asingle complete assay are large.

In an exemplary embodiment of the invention, an iterative measurementuses more flexible gauges (explained below) in a first set ofmeasurement than in a second set of measurements. Alternatively oradditionally, a different subset of gauges is used for the differentsets of measurement.

The difference between the stages may be in correctness of thereconstruction, for example, which interaction locations lie where.Alternatively or additionally, the difference may be in accuracy, forexample, in the distance between two binding locations or the bond angleof an interaction location. In an exemplary embodiment of the invention,the above assumptions of range coverage, for example, for hydrophobicbond sizes and for directional bonds are made stricter in laterreconstruction iterations, for example, providing 15 directional bonds.However, not all the measurements may need to be redone. Instead, onlythose gauges that bond to interaction locations that are expected tochange in the model, are used. Various search methods known in the artmay be used to assist in providing and/or determining convergence of theassay and reconstruction process, for example, hill-climbing.

13. Gauges, Physical Properties

13.1 Overview

Various uses of gauges are described above, some of which may use acomplete gauge library (e.g., completely spanning and having sufficientresolution) and some which may, alternatively or additionally, use apartial library. One or more of several issues are optionally consideredin the design of such libraries. Exemplary such issues andconsiderations that may optionally be used when designing and/orselecting gauges, gauge designs and/or gauges sets are described below.It is noted that some of the issues relate to the properties of theindividual gauges and some to the properties of the gauges as a set. Thedesign (and/or selection) of a complete set of gauges may addressmultiple issues and various tradeoffs, for example as shown in theexemplary gauge set described below. These issues are explored below. Ingeneral, it should be noted that even some of the gauges in a gauge setare not useful, this does not generally detract from the usefulness ofthe gauge set as a whole.

FIG. 4A showed an exemplary gauge 400. A typical gauge set includes alarge plurality of gauges. Possibly, all the gauges share a basic commondesign, as will be described below, however this is not essential. Inaddition, there can be many gauges, gauge designs and gauge sets thatare useful for measurement.

In an exemplary embodiment of the invention, a significant portion of agauge set is based on permutations of a small number of basic molecules,called scaffolds. In this design method; a scaffold includes a pluralityof attachment points and each gauge is created by selecting a scaffoldand mounting various moieties at the attachment points. One potentialbenefit of this approach is that fewer different chemical processes arerequired for synthesizing a library. Another potential benefit is thatthe generated library has more predictable chemical behavior, reflected,for example in the environments used for assaying. Another potentialbenefit is that a more predictable and/or controlled set of distancesbetween moieties may be achieved. Another potential benefit issimplicity is designing a spanning library. Another potential benefit isthat it is easier to ensure spanning in a library or library portion.Another potential benefit is using this type of permutations (possiblywith scaffolds novel to the library) supports generation of missing ordesired measures, ad-hoc. In one case, for example, new gauges withparticular distances are generated by modifying an existing scaffold. Itshould be noted that not all these potential advantages are expected inevery embodiment of the invention.

It should be appreciated that for a given library, parts may be based onscaffolds, while other parts are generated using other means, forexample, selection form an existing molecular library and/or constructedusing various molecular construction, design and synthesis methods knownin the art for attempting to custom create molecules with certainproperties. Further, the entire library can be non-scaffold based. Itshould also be appreciated that not all scaffold-based libraries provideall, some or even any of the above potential benefits.

13.2 Scaffold

In FIG. 4A, gauge 400 is shown to include a scaffold 402, to which fourmoieties are attached, at four of possibly more potential attachmentpoints. In an exemplary embodiment of the invention, gauges 400 areselected to span a range of distances between moieties. In an exemplaryembodiment of the invention, by varying the locations of connection ofmoieties among available attachment points, different inter-moietydistances are fixed for a single scaffold. A greater range of possiblevalues is optionally achieved by providing a range of possiblescaffolds. It should be noted however, that no scaffold is required, perse. Rather, it is expected that at least for some embodiments of theinvention, it may be more cost effective to create a librarycombinatoricly using scaffolds. This is exemplified in FIG. 4B, Wherethe gauge is shown as a triangle defined by its moieties and thedistance between them, without any reference to the scaffolding.

However, in an exemplary embodiment of the invention, a scaffold isprovided on which multiple different gauges are constructed. A pluralityof different or same moieties may be selectively attached to differentlocations on the scaffold, using relatively standardized methods ofcombinatorial-chemistry, thus creating a range of gauges, possiblyhaving generally known chemical properties (e.g., solvency, vaporpressure, stability).

In some embodiments of the invention, the scaffold(s) is selected sothat it does not extend to or out of the triangle shape(s) defined bythe moieties. Alternatively or in some cases, the scaffold and/or someof the moieties do interfere with the binding, and may cause stericclashes. By providing a range of scaffolds, steric clashes may beavoided for some gauges and/or the causes of the steric clashes may bedetermined.

In some embodiments of the invention, the scaffold geometry and/orchemistry is meaningful.

Optionally, the participation of the scaffold in the provision ofbinding triangles is ignored in the design of the gauge set.Alternatively, the scaffold chemical activity is noted during the designof the set, for example, for providing one or more moieties. Optionally,the effect of the scaffold on providing binding, repelling and/orinterfering bonds, is considered during reconstruction or analysis.Alternatively or additionally, the geometry of the scaffold is takeninto account during analysis, e.g., to determine causes for stericclashes.

Alternatively or additionally, triangle binding analysis ignores anybinding triangles that are probably not exposed to the target (e.g.,based on gauge geometry).

13.3 Volumetric Geometry of Gauges

Triangles, as a rule, define a plane, which may or may not be the planeof the scaffold (if any). In an exemplary embodiment of the invention,when gauges are selected for inclusion in a library they are selected sothat their attached moieties lie in a plane or in some other desirableconformity. A planar arrangement has a potential advantage of preventingmulti-stable (e.g., conformal changing) molecules from being included,which is not desirable in some embodiments of the invention, as they mayconfuse the analysis and/or reduce the binding probabilities. Possibly,a set of gauges is provided, to cover a range of possible non-planarorientations. In some embodiments this is more desirable than selectinga molecule that exhibits conformal changes. Molecules with conformalchanges may be excluded using other methods as well, for example, byanalyzing each potential gauge. Alternatively or additionally, thegauges are selected so that the dimensions of the gauge or of particulartriangles in it do not change, even if other parts of the gauge exhibitconformal changes. Optionally, a certain triangle in a gauge may beneutralized by making it energetically unlikely to bind, for example, byensuring that that triangle exhibits conformal changes or addingflexibility to the bonds of one or more of its moieties. It should benoted however, that such exact modification of a gauge may not bepossible, for example, due to the small size of a gauge or its possibleeffect on other parts of the gauges and/or other triangles.

13.4 Flexibility

The flexibility of a gauge can adversely affect one or both of theamount of information provided by the gauges matching and the affinityof the gauge to the target. While it is true that flexible molecules aremore likely to find an arrangement of points to bind to, increasedflexibility may, at least in some cases, reduce the overall probabilityof binding of a molecule, for entropic reasons. In addition, the bindingof a flexible molecule provides less precise information than thebinding of a rigid molecule.

Thus, although a greater number of interaction location layouts can bematched using a flexible gauge, in an exemplary embodiment of theinvention, at least some relatively rigid gauges are selected for thegauge library, so that the measurements using these gauges are moreprecise. Optionally, substantially all gauges in a gauge set aresubstantially rigid. In an exemplary embodiment of the invention, thegauges are translationally rigid, in that the distance between moietiesdoes not change much. Alternatively or additionally, the gauges arerotationally rigid, in that the relative orientation of the moietiesdoes not change. Optionally, flexibility extends to chemical specificityof the moieties, for example, by selecting moieties that are either moreor less specific. For example, one can chose moieties that have only onefunction (i.e., for hydrophobic chose tert-butil or a non-aromatic ring(e.g. cyclohexane) or for hydrogen bonds avoid using a hydroxyl (OH)(which is both a donor and acceptor), or vice versa.

In an exemplary embodiment of the invention, however, a small degree offlexibility is provided, for example to ensure overlap between gauges.In one example, the degree of flexibility is sufficient so that a pairof moieties in the target can be matched by multiple pairs of moietiesin the gauges, with different distances between them. In an exemplaryembodiment of the invention, the gauges are designed such that eachdistance between moieties in the target can be matched both by a gaugethat has a slightly longer distance and by a gauge that has a slightlyshorter distance. The degree of flexibility may be defined so that arelatively low amount of energy is required to bend or stretch the gaugeso that it can match the moiety layout in the target. The relevantenergy levels may depend, for example, on the assay sensitivity, on thegauge concentration and/or the assaying environment.

Optionally, at least a small number of the gauges are flexible, forexample to compensate for gauges that are not available. For example, asnoted herein, rotational flexibility may be allowed for hydrogen bondparticipants and/or aromatic rings. Alternatively or additionally,flexible gauges are used to assist in providing coarse level informationwhich may be later fine-tuned using rigid gauges. Optionally, thereduced amount of information (e.g., by lack of binding and/or lessprecision) is compensated for by the redundancy of the gauges andtriangle measures in the gauges.

It should be noted that particular method of determining which trianglebound, described above, provides a significantly greater weight to rigidtriangles. It should be noted that in a single gauge, triangles may havedifferent rigidities.

In an exemplary embodiment of the invention, the Catalyst software fromAccelrys (formerly MSI) is used to assess the rigidity of a gauge.

In an exemplary embodiment of the invention, at least 20%, 40%, 60%, 80%or any smaller, intermediate or larger percentage of the gauges arerigid. In general, if more rigid gauges are used, they are easier toanalyze using the methods described herein. However, such gauges may notbe available and/or it may be desirable for various reasons to usenon-rigid molecules, for example, if such molecules are similar to drugsor have other properties believed to make them suitable for screening.

In an exemplary embodiment of the invention, a substantially rigidmolecule (or bond) is defined as a molecule which has a single entropicconfiguration and, in which, except for hydrogen atoms, no bond changesby more than 1 Å using less than 20 kCal/Mole. Alternative embodimentsof the invention may allow less rigidity, for example allow greatermovement, such as 0.8 Å, 1.5 Å, 2 Å or any greater, smaller orintermediate value, at 10 kCal/Mole, 15 kCal/Mole, 30 kCal/Mole, 40kCal/Mole or any smaller, intermediate or greater application of energy.It should be appreciated that absolutely rigid molecules are generallynot possible. Instead, the term “substantially rigid” is used in theclaims. As the molecules become less rigid, they may bind with moredifficulty and be less specific in the meaning of their binding.However, less rigid molecules may be easier to obtain and/or use toensure coverage, for example.

Typically, rigid molecules are those for which all single bonds areeither part of a ring or attach “end” atoms i.e. at one of their ends(e.g., single atoms or simple moieties such as NH₂, for which rotationis uninteresting in some cases). Once the ring grows too much, forexample beyond 5 or 6 atoms in some cases, the ring becomes flexible.Larger rings may also be rigid, for example, if there are never morethan 2 adjacent single bonds whose atoms participate only in singlebonds (i.e. if any of the atoms in the ring are themselves attached by adouble bond to an atom that is not a member of the ring, this also mayrigidify that segment of the ring). A single covalent bond isrotationally free, unless it is part of a ring.

13.5 Gauge Lengths

In an exemplary embodiment of the invention, the gauge sides lengths(i.e., the distances between the center of mass of the moieties) areselected to cover a range of expected distances between interactionlocations and/or dimensions of small molecule drugs. Alternatively, forexample, for non-small molecule drugs, a different range may be selectedthan for small molecule drugs. In an exemplary embodiment of theinvention, the selected range is between 2 Å and 12 Å. In anotherexample, the range is to under 10 Å, or under 8 Å. Alternatively oradditionally, the range is from above 3 Å or above 4 Å. In some cases,an “outer length” or an “inner length” may be useful, which are definedfrom the outside or inside of the moieties taking part in a triangle.

In an exemplary embodiment of the invention, the sampling is selected touniformly sample an energy cost required for a molecule to accommodatethe sampling resolution. For example, if a first triangle side is x Åand a second triangle side is y Å, the range of distances covered by thefirst side should require a same amount of energy to modify the moleculeto fit the range, as the range of distances covered by the second side.Generally, this means that as the molecule is larger, the binding range,for a same amount of energy, increases. Optionally, the allowed amountof energy is a parameter of the assaying process, the target and/or thegauges used, for example, to allow a detectable binding by the gauges.

In an exemplary embodiment of the invention, the range is covered byintermediate sizes, so that at least one gauge will match eachintra-moiety distance, for each pair of moieties. Alternatively oradditionally, at least two gauges or gauge sides are similar in moietygeometry. Alternatively, only two gauge sides match. Differentenvironments may dictate a different number of gauges, for example, somebonds may exhibit more flexibility at one temperature, but not atanother.

The sampling of distances by the gauges may be even along the range orit may vary, for example being exponential and/or stepped, due to theeffect of the changing scaffolds between triangles, to achieve differenttriangle side lengths.

It should be noted that some sets of side lengths cannot be combined ina single triangle, due to the required relationship in a triangle,namely, that the sum of lengths of any two sides be greater than thelength of the third side.

13.6 Environmental Stability

In an exemplary embodiment of the invention, the gauges are applied tothe target under normal physiologic conditions, including controlled pH,temperature and ionic content. They may thus be selected to performcorrectly only in the standard environment.

However, in some embodiments, the testing range may not match thephysiological conditions normally present. In a particular example, adesired property of a drug may be activity at hyperthermia temperaturesor for patients with a fever and not at normal physiologicaltemperatures.

A special set of gauges may be used for non-physiological conditions,for example replacing some gauges with others. Alternatively oradditionally, a relatively stable set of gauges may be provided, whichexhibit a same behavior over a wide range of environments. Alternativelyor additionally, even if the gauge properties change, if the change isknown and spanning is retained, the reconstruction method may beadjusted (e.g., the locations and/or amplitudes in triangle space) toaccount for environmental effects.

Another possible environmental variable is the type of solvent used, assome gauges may not be very soluble in water, so assaying may usenon-standard solvents.

In another example, the target may exhibit conformal changes, which aredesired to be measured, under small changes in the environment, such asthe concentration of calcium ions. It may be desirable that the gaugesdo not exhibit the same sensitivity as the target protein to thechanges.

Alternatively or additionally, the gauges may be designed or selected tochange in different environments, thus, for example, allowing a singlegauge to make multiple measurements, each at different environments.

13.7 Uniqueness of Gauges and Overlap of Sides and Triangles

As alluded to above, two different gauge-sides lengths may match aparticular interaction location configuration, for example, by aninteraction location being capable of binding to two different moietiesand/or due to flexibility in the gauges (and/or the target), whichcannot be completely eliminated.

In an exemplary embodiment of the invention, the overlap between gaugemeasurements is controlled to be substantially constant over the gaugespace. Alternatively or additionally, the overlap is minimized.Alternatively, at least a minimum amount of overlap is encouraged, forexample to compensate for various eventualities where a gauges does notbind or an assay fails or to provide additional linking information.

It should be noted that even if substantially rigid gauges are used,there is a level of tolerance inherent in the interaction, so that somefreedom is always available, albeit, possibly at the expense of bindingstrength.

If the degree of overlap is known, its effects can be compensated for inthe above reconstruction method, for example during clustering.Alternatively or additionally, if an expected degree of overlap does notexhibit expected effects, the measurement is suspect.

In an exemplary embodiment of the invention, however, a large degree ofoverlap is provided, for example a factor of two, three or morerepetition of triangles. Fractional overlap may be provided, forexample, by using moieties that have non-orthogonal affinities (in thedetectable range) and/or, as a result of partial overlapping betweentriangles. Generally however, an exactly same triangle will not berepeated, for example, due to differences between scaffoldings and/oreffect of other moieties within a scaffolding.

Thus, alternatively or additionally, to accidental overlap, some or alltriangles are repeated between gauges. In an exemplary embodiment of theinvention, this repetition is used to compensate for the effect ofsteric clashes and/or other unexpected chemical behavior exhibited bysome of the gauges. Alternatively or additionally, the repetition isprovided to assist in determining which triangle bound, based on thebinding of gauges. To this effect, the gauges may be selected so thatthere is a lesser overlap between gauges with respect to the othertriangles the two gauges include. It appears, however, that if thescaffolds are sufficiently different, the probability of most of thetriangles in one scaffold overlapping with most of the triangles inanother scaffold is small. This may assist in distributing theoverlapping between different scaffolds and gauges. Alternatively,similar scaffolds may be used, so that a greater degree of overlappingof triangles of same gauges may be provided. It should be noted thatpart of the overlap is provided by the fact that the gauges may havesome degree of flexibility, so a same triangular array of binding pointscan be matched by triangles of different sizes. In one exemplaryembodiment of the invention, the library is designed so each triangulararray of points can be matched by at least one larger triangle and atleast one smaller triangle. This overlap may be in addition or insteadof repetitive type overlap where a substantially same triangle isprovided at least twice.

Optionally, the order of moieties in a particular scaffold is controlledto account for expected steric clashes, for example, to assure that atleast some triangles will not have the same steric clash problems asother triangles.

Alternatively or additionally, a mixture of gauges, having sametriangles, but different expected steric clashes may be mixed in asingle assay, to help avoid the steric clash problem.

In an exemplary embodiment of the invention, while triangle overlap ingeneral and are not exactly the same, the gauge triangles of at leastsome of the library, for example, 20%, 40% 60% or any smallerintermediate or larger percentage, are selected so that distribution oftriangles in triangle space forms a relatively discrete grid, withclusters of triangles near grid points. Alternatively, at least some ofthe library, for example, 20%, 40%, 60% or any smaller, intermediate orlarger percentage, is selected so that the coverage of the trianglespace is relatively uniform, with less clustering. As noted above,overlap may be useful to overcome various causes of non-binding.However, greater overlap may mean a larger library.

It should be noted that overlap degree need not be uniform. For example,certain triangle sizes may be more prone to steric clashes (e.g., ifthey all use large scaffolds), in which case a greater overlap may beprovided. Optionally, the clustering methods take the degree of overlapinto account, for example to determine a threshold for deciding if atriangle was bound.

13.8 Gauge Mass and Size

In an exemplary embodiment of the invention, the gauges are selected tohave a minimal mass. It is expected that as mass increases, a gauge ismore energetic and less likely to bind. Alternatively or additionally,greater mass often means greater size and more chance for stericclashes. In an exemplary embodiment of the invention, the scaffolds areselected to have a mass under 200, not including moieties. Possibly, theincreases mass of benzene ring moieties is offset, at least in part bytheir enhanced affinity. Alternatively or additionally, gauges areselected by size, for example to be no larger than 4 fusen rings in size(e.g., about 10 Å). Alternatively or additionally, when selecting amolecule for inclusion as a gauge, the selection is failed if themolecule is too large or too massive. It should be noted that in somecase, the size considerations are relative. For example, it is desirablein some embodiments of the invention that a triangle have sides on theorder of a size of a scaffold. Small triangles on a large scaffold maybe ignored when considering the triangles contributed by a particulargauge, and possibly forced to be provided by a smaller scaffold.

It should be appreciated that these examples are not limiting and agauge may be larger and/or have a greater mass or be limited to besmaller and/or have a smaller mass, depending on the application orimplementation, for example.

14. Particular and General Gauge Set Design

14.1 Example Spanning Library Size

Under certain assumptions, the following is an estimation of the numberof gauges and triangles in a complete spanning library for smallmolecules on protein targets.

Assuming the range of lengths to be covered is 9 Å, at steps of 1 Å, thenumber of possible triangles is 10*10*10/(2*3) (factor of 2 for trianglein equality and factor of 3 for rotational degeneracy. Assuming 10moieties and moiety directions, gives about 166,000 triangles. Assumingan overlap factor of 3 and 5 triangles per gauge, gives about 100,000gauges. These numbers are of course only exemplary, but may serve toclarify the following description of library design.

It can be seen that the size of the library depends on the trianglespace to be spanned, the degree of accuracy, complexity of gauges andthe degree of overlap. Any of these may be varied in accordance withexemplary embodiments of the invention, for example, yielding librarieswith between 10,000 or fewer gauges and 1,000,000 or more gauges.Exemplary intermediate library sizes include 30,000, 60,000, 80,000,200,000 and 550,000 gauges. In addition a library may include non-gaugeelements or may form part of a much larger screening library, forexample as described above. In general, the more gauges in a library themore work it is to apply as a whole. However, greater accuracy,specificity and coverage may be available as the library size increases.

An example of smaller gauge libraries, are those that have only 7moieties, reduce the sampling distance to 8 and/or reduce the overlapfactor to 2. Smaller and larger libraries and/or other modifications oflibrary parameters, can also be provided in some embodiments of theinvention, as well as various partial libraries.

In another example, all gauges are designed to include a single triangle(or a small number), in which case about 166,000 gauges are needed (ifthere is no overlap). In such a specific-gauge library, the initialclustering step is optionally omitted. However, it is noted that gaugeswill generally include, at least inadvertently, more than one measure,so that clustering may still be useful. In some cases, a moiety isprovided on a gauge to prevent the scaffold part of the gauge fromparticipating as part of a measure and/or to reduce the number ofdifferent triangles provided by a particular gauge.

14.2 Gauge Subset Selection

A particular type of gauge library is a subset library, which may besmaller than a standard library (but it may be larger, for example, ifit has a higher resolution of lengths and/or moiety types).

In an exemplary embodiment of the invention, only a subset of all thegauges are used for a particular measurement. In some cases this isbecause of the use of an iterative approach, which does not use all theavailable gauges at every step. Alternatively or additionally, it may bedesired to reduce the number of assays performed. Alternatively oradditionally, this may be the result of a large overlap betweendifferent gauges. In an exemplary embodiment of the invention, gaugesare selected to better operate in an environment (e.g., temperature, pH,solvent used) and/or exhibit fewer adverse interactions with the targetand/or the assay, for example, in a cellular assay. Alternatively oradditionally, this may be the result of a failure to create a completespanning library, for example as shown in the example above which may benearly universally useful for all protein targets of small drugs.

It should be noted that one potential advantage of rigid gauges is thatthe geometry of many rigid molecules is minimally affected byenvironmental changes, even if their chemical behavior is affected. Thismay allow the gauge set to be more universal.

In an exemplary embodiment of the invention, gauges for the subset areselected based on the target type, for example, the expected range ofdistances between the interaction locations.

Alternatively or additionally, the gauges are selected responsive to ameasurement need. For example, if a certain interaction location has anunknown size but is known to have a weak affinity, a denser sampling ofthe moiety size range may be used for that interaction location (e.g.,for gauges that are expected to bind to that location).

Alternatively or additionally, the gauges are selected responsive toknowledge of the available drug types, for example, the types ofpossible hydrogen bond directions in the drug. Alternatively oradditionally, the gauges are selected to better distinguish between twopotential drugs, by providing better resolution for the differencesbetween the drugs.

In some embodiments of the invention, the gauges are selected so that anapproximately correct model can be reconstructed, even for those partsof the target for which lower resolution gauges are used. Alternatively,the gauges are selected to determine if a certain drug should bind tothe target, so only gauges required for measuring a smaller range ofpossible configurations are necessary.

Optionally, the gauges are selected responsive to a desired type of bondmatching, for example, if the target and/or potential drug is known toinclude sulfate bonds, gauges including sulfate moieties are used.

In an exemplary embodiment of the invention, a method of selecting agauge subset comprises:

-   -   (a) determining a use of the gauge subset;    -   (b) determining a rule or rules for selection of gauges to meet        said use (e.g., sizes, moieties, densities, etc., e.g., as        above);    -   (c) selecting from the library a plurality of gauges that meet        said rule(s); and    -   (d) optionally, determining if the resulting library is likely        to provide the desired information for said use. For example, a        simulation may be made to see if the assay results are likely to        result in a reconstruction (e.g., based on assay binding rate,        density of coverage, properties or target and/or degree of        overlap required to distinguish between triangles on a gauge).        In another example, the information is partial information and a        simulation is run to see if the information can be        distinguished.        14.3 Gauge Library Design

The following table shows an exemplary set of scaffolds for a gaugelibrary design: TABLE I

AutoNom Name: Thiophene

AutaNom Name: 1H-Pyrrole

AutaNom Name: Furan

AutaNom Name: Benzene

AutoNom Name: Pyridine

AutoNom Name: Pyrimidine

AutaNom Name: Pyrazine

AutoNom Name: 6H-Thieno[2,3-b]pyrrole

AutoNom Name: 1,6-Dihydro-pyrrolo[2,3-b]pyrrole

AutoNom Name: 1H-Indole

AutoNom Name: Thieno[2,3-d]pyrimidine

AutoNom Name: 6,7-Dihydro-pyrazolo [1,5-a]pyrimidine

AutoNom Name: Ouinoline

AutoNom Name: Isoquinoline

AutoNom Name: Quinoxaline

AutoNom Name: 3,4-Dihydro-benzo[e] [1,4]diazepin-5-one

AutoNom Name: 3,8-Dihydro-4H-pyrrolo [2,3-e][1,4]diazepin-5-one

AutoNom Name: 3,4-Dihydro-thieno[2,3-e] [1,4]diazepin-5-one

AutoNom Name: 3,6-Dihydro-4H-pyrrolo[3,2-e] [1,4]diazepin-5-one

AutoNom Name: 5H,11H-Dibenzo[b,f][1.5] diazocine-6,12-dione

AutoNom Name: 1,4-Dihydro-10H-1,4,10-triaza-benzo[a]cyclopenta[e]cyclooctene-5,11-dione

AutoNam Name: 4H,1OH-1-Thia-4,10-diaza-benzo[a]cyclopenta[e]cyclooctene-5,11-dione

AutoNom Name: Dipyrrolo[1,2-c;2′,1′-e] Imidazol-5-one

AutoNom Name: 1,4,7,9-Tetrahydro-1,4,6,9-tetraaza-dicyclopenta[a,e]cyclooctene-5,10-dione

AutoNom Name: 4,7,9-Trihydro-1-thia-4,6,9-triaza-dicyclopenta(a,e)cyclooctene-5,10-dione

AutoNom Name: 2,4,9-Trihydro-1lambda*4*,6,dlthia-4,9-diaza-dicyclopenta[a.e]cyclooctene-5,10-dione

AutoNom Name: 6,9-Dihydro-5H-1-thia-5,8,9-triaza-cyclopenta[a]azulen-4-one

AutoNom Name: 3,10-Dihydro-4H-[1,4]diazepino [5,6-b]indol-5-one

AutoNom Name: 3,6-Dihydro-4H-[1.4]diazepino [6,5-b]indol-5-one

AutoNom Name: 7,8-Dihydro-1H-1,7,10-triaza-cyclohepta[e] inden-6-one

AutoNom Name: 8,9-Dihydro-3H-3,6,9-triaza-cyclohepta[e]inden-10-one

AutoNom Name: 7,8-Dihydro-1H-1,5,8-tziaza-cyclohepta[f]inden-9-one

AutoNom Name: 8,9-Dihydro-5,6,9,11-tetraaza-cyclohepta[b]naphthalen-10-one

AutoNom Name: 3,4-Dihydro-[1,4]diazepino[5,6-b]quinolin-5-one

AutoNom Name: 8,9-Dihydro-4,8,11-triaza-cyclohepta[a]naphthalen-7-one

AutoNom Name: 11H-10,11-Diaza- benzo[b]fluorene

AutoNom Name: α-hydroxyacids

AutoNom Name: α-aminoacids

AutoNom Name: cohels

AutoNom Name: Bicyclo[2.2.2]octane

AutoNom Name: 2-Methylene-2.3-dihydro- benzo[1,4]dioxine

AutoNom Name: 6,7-Dihydro-2H-pyrazino [1,2-a]pyrimidine

AutoNom Name: 9H-Fluorene

AutoNom Name: 1,4-Diaza-bicyclo[2.2.2]octane

AutoNom Name: 1-Aza-bicyclo[2.2.2]octane

AutoNom Name: Pyrido[2.3-d]pyrimidine

AutoNom Name: 5-Methylene-1,5-dihydro- pyrrol-2-one

AutoNom Name Benzo[4,5]imidazo [1,2-a]pyrimidine

AutoNome: 1,4-Dihydro-benzo[4,5] imidazo[1,2-a]pyrimidine

AutoNome: 4,10-Dihydro-1,4a,10-triaza- phenanthren-9-one

AutoNom Name: 1,5-Dihyro-imidazo [1,2-a]pyrimidin-2-one

AutoNom Name: 1,2,3,5-Tetrahydro-imidazo [1,2-a]pyrimidine

AutoNom Name: Thiazolo[3,2-a]thleno [2,3-d]pyrimidin-5-one

AutoNom Name: 1,9-Dithia-4a,10-diaza- cyclopenta[b]fluoren-4-one

AutoNom Name: 5,6-Dihydro-1-thia-5,7,8.9a-tetraaza-cyclopenta[e]azulen-4-one

AutoNom Name: 6,10-Dihydro-5H-1-thia-5,7,10a-tria- benzo[e]azulen-4-one

AutoNom Name: 4,5,-dihydo-3-thia-4,5a,10-triaza-cyclopenta [a]fluoren

AutoNom Name: 8H-1-Thia-cyclopenta [a]indene

AutoNom Name: 3-Thia-4,5a,10-triaza- cyclopenta[a[fluorene

AutoNom Name: 6,7,9,11-Tetrahydro-10-thia-6,9-diaza-indeno[1,2-a]azulene-5,8-dione

AutoNom Name: 2,3,8,7.12,12a-Hexahydro- pyrazino[1′,2′:1.6]pyrido[3,4-b]indole-1,4-dione

5,10-Dihydro-4H-2,3a,10- triaza-cyclopenta[a]fluorene

AutoNom Name: 5H-Pyrido[4,3-b]indole

AutoNom Name: 11H-indolizino[1,2-b] quinolin-9-one

AutoNom Name: 1,2-Dihydro-2-4a,9-triaza- anthracene-3,10-dione

AutoNom Name: 6H-Isoindaol[2,1-a]indole

AutoNom Name: 1,5-Dihydro-benzo[b] [1,4]diazepin-2-one

AutoNom Name: 5,10-Dihydro-dlbenzo [b,e][1,4]diazepin-11-one

AutoNom Name: 5,11-Dihydro-benzo[e]pyrido [3,2-b][1,4]diazepin-6-one

AutoNom Name: 4,9-Dihydro-3-thia-4,9-diaza- benzo[f]azulen-10-one

AutoNom Name: Benzo[g] quinoxaline

AutoNom Name: Pyrazino[2,3-b] quinoxaline AutoNom Name:Pyrido[2,1-b]quinazolin-11-one

AutoNom Name: 1-Thia-4a,9-diaza-cyclopenta [b]naphthalen-4-one

AutoNom Name: 2-Methylene-4H-benzo[1,4] thiazin-3-one

In an exemplary embodiment of the invention, the moieties are Me(methyl), Et (eteyl), Pr (propyl), Ph (phenol), CO₂H, OH and NH₂.Although the moieties may be connected at any of the R locations, notall the possible gauges are needed, as explained above. The indolizinescaffold can have, at R1, either COOH or NH₂, both of which are shown inthe table. In particular, applicants have found that in general, ascaffold with four or five attachment points can spa n its entire rangeof triangles with M moieties, using only about M³ different gauges. Thisis believed to be generally true (e.g., the exponent is not much higherthan 3) for scaffolds with a larger number of attachment points.

It should be noted that even if a library does not cover all thepossible triangles, a viable reconstruction is still possible for manydrug targets and/or considerable utility attached to the library. Also,as noted above, partial reconstruction is useful in some cases. Also, asnoted above, gauge matching can be used as leads and/or to reject leads,even if no reconstruction is possible, in some cases. In someembodiments of the invention, a failure of the method is typicallyself-evident and does not create an unproductive search afternon-existent leads.

Alternatively to constructing a library of gauges from scratch, at leastpart of the library can be generated by scanning existing libraries formolecules that include triangles having desired sizes and/or moieties.Optionally, molecules that are small and rigid are selected, asdescribed above. This type of library, for example, may not be based ona set of scaffolds.

14.4 Library Building Method

From the above description, it should be clear that there are manymethods that may be used to construct a library. The following exemplarymethod is described, at least partly to illustrate various applicationsof the above rules:

-   -   (a) determine library parameters: e.g., spanning range and        accuracy desired for library;    -   (b) select moieties for library;    -   (c) select a scaffold;    -   (d) generate gauges from the scaffold;    -   (e) add generated gauges if they are suitable;    -   (f) repeat (c)-(e) until the library spans the range with a        desired accuracy and/or coverage; and    -   (g) optionally, check library.

In accordance with example embodiments of the invention, a resourceallocation algorithm is used, for example the greedy method or the firstfit method. These names refer to methods of selecting from a set ofpossible resources, which resource to allocate at a particular time, forexample, which gauge to choose for a library from available gauges on ascaffold or which scaffold to add to the library. Many such methods areknown in the art and may be used, noting that the method is notrequired, in some embodiments of the invention, to provide an optimalsolution, just a working or reasonable solution.

An alternative method is a selection-based library construction method.In this method, existing molecule libraries are scanned for moleculesthat have gauge-like properties (e.g., as described herein). Theresulting potential gauges may be filtered out to remove redundancies.It is expected however, that in the current state of public libraries,scanning such libraries will not yield a complete gauge library.Optionally, such a selected gauge library will be completed using othertechniques, such as scaffold based gauge generation.

It should be appreciated that given a large number of possible gaugesand a smaller actual required number, there are many optimizationtechniques for selecting a suitable and/or optimal set of gauges thatmeet the required number. As noted above, the selection may be based onthe use to which the library is put and/or be based on considerationssuch as diversity, chemical behavior and ability to synthesize. Inaddition, a part of a library may be replaced, for example with a set ofgauges constructed from other scaffolds or using molecules selected froma library of potential leads. In an exemplary embodiment of theinvention, at (g) a constructed library is optimized, for example,removing redundancies and ensuring that desired distributions (e.g., oftriangles, chemical properties) and overlaps (e.g., of lengths and/ormoieties) meet certain guidelines and/or are optimal.

14.5 Scaffold Selection Method

In an exemplary embodiment of the invention, scaffolds in general areselected to have certain desirable properties, for example, one or moreof:

-   -   (a) small size;    -   (b) rigidity;    -   (c) suitability for combinatorial chemistry;    -   (d) including a plurality of attachment points, for example, 3,        4, 6, 10, 12 or any smaller intermediate or larger number, for        attaching moieties and/or chemical markers (e.g., for binding        assays, chemical manipulation);    -   (e) a geometric arrangement of the attachment points so that a        range of triangle sides can be provided;    -   (f) 3D structure, for example planar or volumetric may be        preferred for different situations;    -   (g) number of excess protrusions (in some cases may be desirable        to be small), to which moieties may or may not be attached, so        that excess is relative to a perfect scaffold where the useful        (e.g., for the library or for a particular triangle) moieties        define the shape of the scaffold; and/or    -   (h) solubility (may be determined, for example, based on the        number of polar atoms in the scaffold).

In general, as more attachment points for moieties are provided, thescaffold is more able to provide triangles of various sizes, however,this may adversely affect the scaffold (and gauge size) and many of thetriangles may be useless. In a scaffold in general, it may be useful todesignate only some of the potential attachment points as attachmentpoints to be used. This may reduce the number of different synthesismethods used and/or promote uniformity thereof.

Not all or even any of these properties are essential in someembodiments of the invention. As a practical matter, small rings andring chains appear to meet these criteria. Thus, in an exemplaryembodiment of the invention, a set of scaffolds may be generated byreviewing existing known rings and small chains for molecules that meetthe desired criteria. In an exemplary embodiment of the invention,during this type of selection an effort is made to select scaffoldshaving a range of sizes (e.g., distances between attachment points), sothat a range of triangles may be generated using the scaffolds.

In addition to scaffold criteria in general, a selection of scaffoldsfor a library may impose other criteria, for example that the scaffoldsgenerate a spanning library of gauges and/or a range of chemistriesand/or require a relatively small number of relatively low complexityprocess to generate the gauges.

In an exemplary embodiment of the invention, the scaffold selectionprocess is as follows. Given an existing library portion, a new scaffoldis selected from a list of available potential scaffolds if it answersat least one of the following criteria:

-   -   (a) the scaffold generates a large number of triangles that are        missing from the libraries, for example, 10, 50, 100 or any        smaller intermediate or larger number, such as a user set        number;    -   (b) the scaffold generates at least one (or a small number of        triangles, such as less than 20, less than 10 or less than 5, or        any other user set value) triangles that have evaded generation        using other scaffolds and form missing portions of the library,    -   (c) the scaffold has a significant amount of known chemistry        (e.g., methods for manipulation and/or adding moieties); and    -   (d) the scaffold adds the potential for a desired amount of        overlap.

In general, if a larger the number of gauges is produced, it may beeasier to complete a library. However, not all scaffolds can generatelarge numbers useful triangles.

It should be noted that in some divergence based methods of librarydesign, each library element is selected to be as different as possible,so that this type of selection methods and/or at least some of thecriteria used cannot be applied and run against conventional ideas.

It should be noted that as the library fills up, consideration (b) maybe given more weight, with the possibility of searching or constructinga scaffold that has the desired properties (e.g., to form requiredtriangles). Further, the search may lead to selection of less rigidscaffolds, for example, to ensure coverage or due to lack of suitablemore rigid scaffolds.

In an exemplary embodiment of the invention, during an optionaloptimization stage of the library, scaffolds are assessed as to theirquality (e.g., meeting scaffold criteria), number of triangles generatedand/or uniqueness of triangles generated. A scaffold may be removed fromthe library if it is determined to be less useful or unneeded based onone or more of these considerations.

One difference between scaffolds is the number of rings in a scaffold.In general, as the number of rings increases, so does the scaffold sizeand weight. For some applications, the number of rings in a scaffold maybe used as a heuristic to determine what approximate triangle sizes thescaffold can provide. For some applications, multi-ring scaffolds may benecessary. Alternatively or additionally, single or bi-ring scaffoldsmay be useful for small triangle sand/or for reducing steric clashes.

14.6 Gauge Selection Method

In an exemplary embodiment of the invention, gauges in general areselected to have certain desirable properties, for example, one or moreof:

-   -   (a) small size;    -   (b) large numbers of triangles;    -   (c) high or otherwise desirable binding affinity, for example in        the range of 1-100 micro Molar;    -   (d) rigidity;    -   (e) the attached moieties defining the volume of the molecule;    -   (f) relatively uniform binding probability for all moieties, for        example a factor of ten between moieties and a factor of 100        between molecules in a library, however, in other embodiments        other, smaller or greater factors (e.g., ˜1, 5, 20, 50, 130,        250, 1000 or any smaller, intermediate or greater factor) may be        provided for one or both criteria; and/or    -   (g) chemical behavior, such as (i) solubility, for example in a        natural solute of the target (or an approximation thereof), for        example water at a given pH, with some detergent such as DMSO to        aid solubility, (ii) lack of reactivity with expected        contaminants, (iii) lack of chemical reactivity (creation of        covalent bonds) with a target protein i.e., with amino acids or        known typical combinations of them and/or with a substrate, (iv)        desired behavior over a range of properties.

In general, a higher uniformity of binding means that the assays have asame meaning. However, it is generally not practical to provide suchnarrowly defined materials, and a certain latitude is useful if arealistic set of chemical is to be provided.

When generating a library (or part thereof) by selection of gauges fromexisting molecule screening libraries, each molecule is, for examplescreened against the desired criteria. A molecule may be selected orrejected. Alternatively or additionally, a molecule may have a score ofsuitability associated with it. Similarly, a set of potential gauges maybe generated from scaffolds.

In an exemplary embodiment of the invention, gauges are selected fromthe generated/selected set, based on one or both of suitability (e.g.,relative or absolute) and meeting of group criteria. In an exemplaryembodiment of the invention, one or more of the following group criteriaare applied, for example as binary criteria or as part of a score:

(a) That uniqueness of the triangles provided and/or them matchingmissing triangles.

(b) Matching of flexibility of the gauges and/or individual triangles,to desired flexibility.

(c) Shape of gauge as a whole, for example, being elongate or beinground. The shape may be a consideration, for example when building alibrary in which shapes are varied so that steric-clashes will notreject all of a certain triangle. To this end, the shape of the gaugemay interact with the location of specific triangle son the gauge, e.g.,if a same triangle is found on two elongate gauges, it may be desirablethat on one of the gauges the triangle is in an axial direction and inthe other, in a trans-axial direction. Alternatively or additionally,shape considerations relates to the three-dimensional shape of the gaugeand/or relative layout of triangles in the gauge.

(d) That certain non-triangle measures are found, for example specificnon-triangle measures or that a uniform (or other) distribution of such4-5- or other multi-point measures are provided.

It should be noted that for gauges and/or scaffolds, the determinationof suitability may include, for example one or more of using simulationand molecular analysis software, chemical laboratory testing and/orsearching literature for the same or similar chemicals.

The above selection method may be useful when designing a singleuniversal library (or a set of such libraries for broad uses). It shouldbe noted however, that some, similar or other selection methods may beused when generating personal and/or ad-hoc libraries, searching forgauges or measures with particular properties and/or when defining agauge and/or scaffold to be generated.

14.7 Gauge Synthesis

The generation of a gauge library from scaffolds, in some embodiments ofthe invention, may assist in the serial synthesis of the gauges. Inlibraries that are not (or are partially not) scaffold based, standardsynthesis methods may be used.

In an exemplary embodiment of the invention, the gauges are synthesized,for example using liquid phase methods as described below, andimpurities are removed using standard methods, for example using HPLC.

In an exemplary embodiment of the invention, a parallel synthesis methodis used, in which a plurality of gauges are synthesized at once and thenseparated. It should be noted that in some embodiments of the invention,only a small number of the gauges that can be created by a scaffold areactually needed. Alternatively or additionally, even if many of theparticular gauges cannot be created, a sufficient number of alternativegauges may be available, to provide spanning and/or overlap of a desiredtriangle space. For example, on a five point scaffold with 10 moieties,100,00 combinations are possible, of which 1000 are sufficient cover allthe triangles. Thus the choosing can be, for example, ad hoc, such asbased on the actual yield (e.g., relative yield) or based on the priordesign of the library.

In an exemplary embodiment of the invention, combinatorial chemistrymethods are used to attach moieties, each at a different attachmentpoint of a scaffold, optionally so that all combination of moieties arecreated. Each final compound is made attached to a polymer bead (forexample) for ease of separation. The beads may be color coded forassistance in separation and/or identification of the created gauge.

Alternatively, other solid phase methods, for example as described belowor as known in the art, are used.

14.8 Mixed Library Design

As noted above, in order to be useful, a complete universal library isnot required. Further, a gauge library may be included into a “regular”screening library. In an exemplary embodiment of the invention, at least0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 40% or any smaller, intermediate orlarger percentage of the molecules in a library used for screening,measuring and/or other uses comprise gauge-like molecules. Of suchgauges, for example, less than 50%, or greater than 30%, 60%, 80%, 90%,or any smaller, intermediate or larger percentage of the gauges arescaffold-based gauges, where a scaffold is used to generate at least 5gauges with less than 20% overlap in triangles defined by attachedmoieties. As noted above, while a library may include standard screeningparts, providing significant numbers of gauge-like molecules may assistin applying the methods described herein.

In an exemplary embodiment of the invention, the library comprises atleast 5,000, 10,000, 20,000, 50,000, 80,000, 100,000 or any intermediateor greater number of gauges. These gauges may be, for example, scaffoldbased gauges, plain gauges and/or rigid gauges. These gauges may span,for example, 5%, 20%, 40%, 80%, 100% or any smaller, intermediate orgreater percentage of the triangle space, for example, with an overlapof 1.1, 1.5, 2, 3 or any smaller, intermediate or greater degree. Asnoted above, when spanning is better, the degree of success may behigher, albeit at a cost of using a larger library. Smaller librariesmay be easier to apply and still yield useful results, in many cases.

One significant difference between gauges and other lead libraries(e.g., diversity based libraries), in accordance with some exemplaryembodiments of the invention, is that a relatively large number ofmatches is expected using gauge based libraries. For example, at least0.01%, 0.05%, 0.1%,0.2%, 0.5%, 1%, 3%, 5%, 10% or any smaller,intermediate or greater percentage of numbers is expected to bind. Thepercentage of binding may depend, for example on the ratio betweengauges and non-gauge leads in a library.

It should be appreciated that these percentages are not mere numbers.Rather, they represent a qualitative difference from libraries wheremore often than not, no leads bind. The greater the probability offinding one or more leads and the greater the number of leads, the morelikely it is that a drug will be found. However, of binding is toolikely, the quality of information provided by the binding may bereduced.

A library may also include a mix of three-point measures and highervalance measures. While any gauge that includes more than three moietiesincludes a high valance measure, in an exemplary embodiment of theinvention, the library is designed to span the higher valance space. Forexample, the library spans at least 0.1%, 0.3%, 0.5% or at least 1% orany smaller intermediate or larger percentage of the space of the highervalance measures. The spanning may be, for example, continuous (e.g.,the whole library at a low resolution or part of the library at a highresolution) or it may be discrete (e.g., isolated parts of the library).In general, higher valance measures may require a very large number, forexample, 20,000,000 for a spanning equivalent to the 100,000 library ofthe triangles, so commercial implementation may depend on theavailability of even more parallel assays than available today.Optionally, the higher valance measures are provided to be moreflexible, so that a lower resolution is required to span the space.

14.9 Ensuring Library Reliability

In an exemplary embodiment of the invention, once a library isconstructed and/or during its construction, various quality assuranceprocesses may be employed. In one example, the library is analyzed toensure that it meets the spanning, overlap and/or accuracy criteria setfor the library. Any missing triangle and/or gauge may be provided atthis point or noted as missing. Alternatively or additionally, moleculeswith low solubility or high toxicity are removed and/or replaced withmolecules exhibiting similar spatial chemical configurations.

In an exemplary embodiment of the invention, feedback from use of thelibrary is used to calibrate the library, reconstruction process and/orto assist in library design.

In an exemplary embodiment of the invention, the theoretical modeling ofthe library is compared to its actual behavior, for example, by runningtest assays against randomly selected targets having a known and/or anunknown structure. Two examples of molecules with known structures arethoroughly mapped proteins and structures constructed from DNA or RNA,with optional attached elements. Optionally, the targets are not randomand are selected to test certain assumption in the theoretical model ofthe library. Alternatively or additionally, the calibration is providedby analysis the results of real uses of the library over time.

In an exemplary embodiment of the invention, one or more of thefollowing data is provided by such analysis:

-   -   (a) assay binding rates for gauges and families (e.g. similar)        gauges;    -   (b) dependency between environmental conditions and binding        rates and/or conformal changes for one or more gauges;    -   (c) Baysian probability of steric clashes between gauges (and        triangles thereof) with overlapping triangles;    -   (d) actual degree of overlap between triangles;    -   (e) dependency between target type and gauge binding; and/or    -   (f) parameter values (e.g., thresholds) for the various        algorithms.

Other properties of the library, for example general rigidity of thegauges and correctness of values in the data bank may also be providedby such or other analysis.

In an exemplary embodiment of the invention, as a result of the abovefindings, the library is amended, for example, by removing redundantgauges and/or searching for gauges to generate the missing triangles.

Alternatively or additionally, as a result of the above findings, latergeneration of libraries and sub-set libraries is modified to take thecalibration information into account, for example in a specific manneras relating to specific gauges and/or in a general manner as it relatesto statistical deviation of the behavior of scaffolds and/or families ofgauges from their appropriate theoretical models and/or as parametersfor such models.

Alternatively or additionally, the reconstruction process is calibrated,for example to better distinguish which triangle matched, the actualcoverage of each triangle, the spatial shape (in triangle space) of amatch and/or the relative binding strength of various triangle measuresand/or gauges.

14.10 Human Interaction During Library Design

The process of designing a library may be automatic, semi-automatic ormanual. In general, when more potential gauges and/or scaffolds areavailable and suitable modeling software is available as well, automateddesigning may be provided, one example of this is once a completelibrary is available, selecting a sub-set may be completely automatic,once the desired parameters are provided. Some of the library may begenerated automatically in any case, for example selection of gaugesfrom existing libraries and/or selection of scaffolds from existinglibraries. The determination of ease of synthesis may be required to bemanual if no earlier information is available. It is noted, however,that in an exemplary embodiment of the invention, the scaffolds arechosen to have known chemical behavior and synthesis paths, so thatattachment of moieties should require little or no research work. Insome cases, however, a human may be required to not only select betweenalternatives but actually to find a particular missing gauge or suggesta scaffold design. It is noted, however, that the mathematicaldescription of the library in accordance with some embodiments of theinvention, assists and may allow complete or nearly complete automaticgeneration of a library using constructive synthesis and/or analysis ofexisting molecules. Possibly, such a library may then be optimized, forexample as described above, possibly manually, especially to assist inproviding an easy to synthesize library.

As noted above, the reconstruction process may be completely automaticor it may include a manual aspect. In general, however, it is expectedthat the high hit rate of binding of gauges will reduce or eliminate anyneed for human intervention, at least in some of the steps of drugdiscovery. Of course, once mapping is completed, a human user maydesired to test the effect of various assumptions, for example, how thereconstructed layout depends on various assumptions made on the targetconformity. Also, in some case a human expert (or an expert system) maybe used to select among alternative or select likely leads, since inmany cases the method will generate a small number of possibilities fromwhich one or two should be selected, failing that costs may be veryhigh.

In an exemplary embodiment of the invention, one point for humanintervention in the drug discovery process is in designing drugcandidates that match a final pharmacophore (e.g., model). It is noted,that various software exists to assist or automate this step. Typicallyhowever (at this point in time), human judgment is better at assessingsynthetic feasibility for complex molecules. If the suggested drugs arecreated by linking together gauges or simple fragments, however,automatic assessment and possibly generation methods, may be reasonable.

15. EXPERIMENTS AND EXAMPLES

15.1 Experiment 1

Some of the above measurement method was testing using the followingexperiment.

In this experiment, known inhibitors of HIV-1 Protease were analyzed todetect a set of triangle measures that should exhibit binding to HIV-1Protease. A set of molecules including the triangle measures wereselected and physically assayed and shown to have the expected bindingto HIV-1 Protease. The results indicate that triangles are a viablegeometrical sub-structure that can be used to measure a target bybinding.

The following entries in the PDB (Protein Data Base) were extracted asstructures of HIV-1 Protease with known, bound, inhibitors: 1ajv 1ajx1dif 1gno 1hbv 1hih 1hos 1hps 1hpv 1hpx 1hsg 1hte 1htf 1htg 1hvi 1hvj1hvk 1hyl 1ohr 1sbg 1upj 2bpv 2bpw 2bpx 2bpy 2bpz 2upj 3tlh 5hvp 7upj.

The structures were super-imposed using the protein as a referenceframe, so that the spatial position and orientation of the inhibitorswas superimposed. The inhibitor molecules were then decomposed intomoieties and those were clustered in space. Strong bonding locationswere identified based on the sane moiety in different molecules bindingto a substantially same binding location in the protease. Confidence inthese locations was increased by verifying that the protein moieties atthose locations were compatible with the inhibitor molecule moieties.

Triplets of the inhibitor moieties at the strong binding locations wereselected as “triangles”. Gauges, for example, of a gauge set asdescribed above, that have those triangles, are expected to bind, or atleast some of them should bind.

The triplets were used as a query input for a search, in ML's ACD-SC(available chemical directory for screening). Molecules that matched thequeries (moieties and size) and the rigidity requirements were selected,as shown in the following table. TABLE II mg for Density 1 mM No.Compound MW (g/ml) Cat. No. in 10 ml 1

276.35 S-83425-4 2.8 2

403.26 1.008 36,667-6 4.00 μl 3

391.35 S-63995-8 3.9 4

408.32 S-84651-1 4.1 5

324.55 S-2210-2 3.2 6

464.56 R-15419-9 4.6 7

445.57 S-22759-5 1.9 8

445.57 S-22675-0 1.9 9

446.17 S-95285-0 4.5 10

412.53 S-9757-9 4.1 11

464.56 R-15449-0 4.6 12

438.57 R-15358-3 4.4 13

436.55 R-15353-2 4.4 14

422.53 R-33994-6 4.2 15

204.16 S-52812-9 2.0 16

298.34 S-6426-3 3.0 17

200.32 0.887 46443-0 2.26 μl 18

234.34 27302-3 2.3 19

280.37 R-22433-2 2.8 20

236.36 44642-4 2.4 21

268.33 S-4228-6 2.7 22

344.38 NRB-01407 3.4 23

377.51 RJC-03605 3.8 24

245.37 JFD-03358 2.5 25

350.31 RJC-03637 3.5 26

408.63 RJC-03257 4.1 27

435.49 JFD-01334 4.4 28

460.32 RJC-02058 4.6 29

456.59 RJC-02951 4.6 30

477.41 BTB-14801 4.8 31

280.29 BTB-11623 2.8 32

295.43 RJC-03631 3.0 33

212.25 RJF-00720 2.1 34

302.41 85,612-6 35

252.23 25,272-7 36

267.54 29,126-9 37

258.12 23,319-6 38

265.94 30,118-3 39

308.34 16,263-9

The molecules numbering up to 33 were expected to exhibit bindingbehavior, due to them including at least one triplet. The moleculesnumbered 34 and up are superficially similar but do not include therequired triangles.

All of molecules were actually assayed and appeared to show activity(effect on HIV-1 Protease) at various concentrations (between 10 and1000 micro-molar). Of these molecules 1-33 about 60% were found to beactive, in particular molecules 7, 9, 23 and 27. Also molecules 34-39were assayed, with no activity shown, as expected.

As noted above, these results appear to indicate that gauges, ingeneral, that have a triangle measure that matches the target layout,should, often enough, bind in a detectable manner.

15.2 Experiment 2

In this experiment, assay results performed by others were used toreconstruct the spatial layout of binding locations, for known moleculesand then compared to the current state of the art.

The NCI maintains a database of molecules that have tested positive foractivity against HIV. 43,000 results (in the October 1999 release) areavailable at “http://dtp.nci.nih.gov”, under “public data”, then“results from AIDS antiviral screen”. From these molecules were selecteda subset that showed at least a moderate level of activity and wererigid enough to allow determination of the spatial position of all theirmoieties. This resulted in fewer than 200 molecules. The moietytriangles in these selected molecules were clustered.

The clustering results showed a good match to the results of experimentI and the triangles of the molecules were found in the PDB structures.

These results appear to indicate that a set of gauges (e.g., themolecules that were tested for HIV) can be used to measure and thenreconstruct an active area.

In addition, these results appear to indicate that at least part of asuitable library may be generated by selecting suitable gauges fromavailable libraries, rather than by construction using scaffolds. Itshould be appreciated that it may not be required to determine thespatial positions of all the moieties, for example only of the moietieswith a high binding affinity. Moieties with low affinities may beremoved, in some cases.

16. Synthesis Book

Following is a synthesis book, arranged in chapters, for some of thescaffolds (and gauges derived from them), shown in table I. A mostimportant aspect of this synthesis is that it illustrates that suitablescaffolds and gauges are available and can be generated using knownchemical processes applied to standard or modified sources and/or bychanging their parameters in an expected manner. The referencesdescribed in this book are incorporated herein by reference. In anycase, the partial library described in the appendix has at least theproperty that is can serve in many cases to provide a partialreconstruction and/or a significant increase in lead matching.

It should be appreciated that the novel materials described in the book,the manipulation methods thereof, synthesis methods thereof and groupsof molecules from this book are also considered to be within the scopeof at least some aspects of the invention, for example, a libraryincluding one, two, four, six, eight or any intermediate number ofscaffolds as described therein. Alternatively or additionally, a libraryin accordance with an exemplary embodiment of the invention, includes atleast 100, 300, 500, 1000, 2000, 4000, 10,000, 20,000 or any smaller,inter-mediate or larger number of gauges from this book. While it isuseful to select gauges from the book, for example by using thescaffolds described therein to span part of the library, this is notrequired.

16.1 Benzenes, Pyrimidines 6-membered Ring Scaffold

The Biginelli dihydropyrimidine synthesis (pathway below) is a promisingmulti component condensation, which involves the one-potcyclocondensation of β-ketoesters 2, aldehydes 3, and ureas 4 providingthe heterocycle 1, which can be oxidized to the corresponding pyrimidinemoiety.

Biginelli-General Multicomponent Approach

Several protocols have been developed for solution phase Biginellireactions' In order to drive the reactions to completion, howevergenerally, an excess of two of the three components 2-4 has often to beemployed, and purification steps are required. The solid phase synthesisprovides the desired dihydropyriridines in good yield and superiorpurity directly after cleavage from the resin² (pathway below):

Another approach for the SP synthesis of highly substituted pyrimidineswas recently published³. In this work the synthesis starts frompolymer-bonded thiouronium salt 5, which undergoes cyclocondensationwith acetylenic ketones 6 to form carboxy pyrimidines 7 (pathway below).

Tetra substituted pyrimidines a can be prepared via a modifiedBigenelli's synthesis as described in the pathway below:

First the imidine functionalities are formed on the acid labile resin toproduce the resin immobilized amidines 23⁴, urea 24 and guanidine 25².Actually, these amidines served as the first Biginelli building block.Next, the addition of the two other Biginelli building blocks, namely 2and 3, to 23-25 leads to generation of dihydropyrimidine scaffolds 20,21and 22^(5,) respectively. The consequent reduction of ketone moieties(NaBH₄, BF₃OEt₂) leads to 14, 15 and 16, which after cleavage (TFA, DCM,1:1) followed by mild oxidation (CAN, CH₃CN) affords the desiredpyrimidines 8, 95 and 10 respectively. The CAN could be removed, afterthe completion of the oxidation, by Solid Phase Extraction (SPE) or bysimple 96 well SePack. Other oxidation reagents such as MnO₂ ⁶,O-chloranil⁷, KMnO₄ ⁸, and CrO₃, AcOH, H₂SO₄ ⁹ can also be used. In caseR3=OMe (when the building block 2 is β-ketoester) dihydropyrimidines 20,21 and 22 undergo hydrolysis of ester (LiOH, THF or 5% alcoholic KOH¹⁰,producing the 4-carboxy dihydropyrimidines 17, 18 and 19 respectively.Following by the same mode as for 8, 9 and 10 (1. TFA, DCM, 1:1; 2. CAN,CH₃CN) 17, 18 and 19 react to give the sub-library of4-carboxy-pyrimidines 11, 12 and 13 respectively. It should be notedthat in case of unsymmetrical 1,3 diketones 2 a mixture of 2 isomers areobtained.

Core Approach Towards Tetrasubstituted Pyrimidines

It was demonstrated¹¹ that dihydropyrimidine 5-carboxylic acid can betransformed into carboxylic azide which in turn undergoes Curtiusrearrangement to give isocyanate. This reaction provide an excess of5-amino dihydropyrimidines A.

Pyrimidines can be prepared by cyclocondensation of amidines with α-βunsaturated ketone. Recently, the researchers have published thesynthetic work¹², in which they describe the utilization of the Wittigreaction in formation of α,β-unsaturated ketones on SP for the synthesisof the various heterocycles. We propose the alternative three-stepsynthesis of pyrimidines a in solution, based on the formation of theα,β-unsaturated ketone building blocks 26 as a key step^(12b-d) isdescribed below:

α-β-unsaturated ketones 26 can be obtained in good yields and purity byWittig reaction of the appropriate aldehyde and the correspondingtriphenylphosphonium bromide 27 with NaOEt at reflux in DMA. Thephosphorus yields 27 are readily available from α-bromo ketones 28 bythe Arbuzov reaction, followed by treatment with a strong base, such asNaOEt. The reaction of ketones 26 with various amidines 23^(12b-d) (FIG.4) affords, the desired tetra-substituted pyrimidine sub library a.

Small sub-libraries b-g having one or more constant functional group onthe six member aromatic ring, are characterized by better solubility.

A series of 2,5,6-trisubstituted-4-oxo-dihydropyrimidines 29 can beprepared by SP using a cyclization-cleavage strategy¹³ from readilyavailable amidines 23 and resin attached α,β-unsaturated carboxylicacids 30¹⁴ (see pathway below). Compound 30 is obtained via coupling ofthe polymer and acyl-chloride 31 (derivetized from commerciallyavailable α,β unsaturated carboxylic acids.

Compounds 29^(13b) can be oxidized (CAN, CH₃CN) to correspondingpyrimidines b.

A solid phase method for the preparation of Knoevenagel condensationproducts from resin bound malonates and malonic acids has potential forthe preparation of hetero- and carbocyclic compounds. (see pathwaybelow)

Malonic acid monoester (see pathway above) are prepared from macroporousWang resin (AgroPore, Argonaut Technologies)¹⁵ by treatment withMeldrum's acids. Conversion of the unsymmetrical ester 34 was achievedby treatment with trifluoroethanol and DIC, followed by Knoevenagelcondensation with the aldehyde in the presence of piperidine acetate togive substituted methylene malonate 33. For the bulk resin preparationof 33 (2-10 g of resin), the Knoevenagel condensations are carried outwith Dean-Stark trap to eliminate water which gave consistently higheryields and faster reaction). Malonates 33 are treated with 10equivalents of the amidine hydrochlorides 23 in dimethylacetamide (DMA)solution, with excess K₂CO₃ to neutralize the HCl amidine salt, at 70°C. for 4-8 h to give resin bound dihydropyrimidones 32. The reagentconsumption progress can be monitored by FTIR observing the adsorptionsof C═N and C═O groups. Oxidation of 32 with 0.2M ceric ammonium nitrate(CAN) in DMA¹⁶ affords resin bound hydroxy-pyrimidines. Cleaving underacidic conditions (TFA/DCM, 1:1, RT, 1-2 h) gives secondary sub-libraryc (The sub-library c exists in its tautomeric form-4-pyrimidone).

The examples for tailor-made synthesis of miscellaneous tetrasubstituted6-atom membered rings are described below.

The amidines 23-25 react in solution with commercially available[bis(methylthio)methylidene]malononitrile 35 (see pathway below) in thepresence of DEEA¹⁷ to give the corresponding methylthiopyrimidines. Thelatter are oxidized with 1.2 equiv. of m-CPBA in DCM or H₂O₂ ¹⁸, to formthe intermediate sulfinyl derivatives 36 which are subjected to aminesubstitution with NH₃ ¹⁹ (dioxane room temperature) leading, afternitrile hydrolysis (TFPA)¹⁰, to the final aminopyrimidines 37. If LiOHis used instead of NH₃ the corresponding hydroxypyrimidines 38²¹ afternitrile are obtained.

A series of various 3,4,5-trisubstituted phenols 39 can be synthesizedin high yields using the “cyclization-cleavage” approach²².

Base catalyzed reactions between α,β-unsaturated ketones and polymerbonded acetonyl groups 42 (see pathway above) result in a tandem Michaeladdition/annulation reaction with concomitant cleavage from the resin toobtain the desired phenols 39 The synthesis initiates using resinprepared from Merrifield resin by coupling with Sodium3-hydroxypyridine, producing higher loading capacity resin 44, which wassuccessfully quarternized by 1-bromopropane-2-one (or 2-bromo 1-phenylpropn-1-ne; 2-bromo 1,2 diphenyl ethanone; 2-bromo-1-phenylbutane-1-one; 3-bromo butan-2-one) to afford poly-pyridinium salt 43.Reaction of 43 with α,β-unsaturated ketones was carried for 16 h, andafter filtration of the resin the library 39 is obtained.

REFERENCES

-   1. Tet, 32, 6937, (1993).-   2. a. P. Wipf, Tet. Lett., 36, 7819, (1995);    -   b. K. Lewandowski, J. Comb. Chem. 1, 105, (1999).-   3. D. Obrecht, Helv. Chem. Acta, 65, (1997).-   4. Chenera, WO 95/16712, 1995.-   5. Compounds 9 and 21 are stable in their carbonyl tautomer;    heterocyclic Chem. 3. (1984).-   6. Pharmazie, 5435, (1999)-   7. J. Heterocyclic Chem. 24, 589, (1987)-   8. J. Heterocyclic Chem. 23, 1821, (1986)-   9. Chem. Abst. 90, 121631y, (1979).-   10. Montash Chem 107 587 (1976).-   11. Tet, 48, 5473, (1992).-   12. a. A. Marzinzik, J. Org. Chem., 63, 723, (1998)    -   b. WO 9815532    -   c. Sib. Khim/Zh. 87, (1991)    -   d. J. Heterocyclic Chem. 24, 1141, (1987)-   13. a. S. Kolodziej, Tet. Lett., 37, 5277, (1996);    -   b. Synthesis, 86, (1985).-   14. a. D. Powers, Tetrahedron, 54, 4085, (1998);    -   b. K. Ito, J. Heterocyclic Chem., 29, 1037, (1992).-   15. a. B. Hamper, Tet. Lett., 40, 4973, (1999);    -   b. C. Chiu, J. Comb. Chem. 1, 73, (1999).-   16. a. M. Gordeev, Tet. Lett., 37, 4643, (1996);    -   b. S. Tadesse, J. Comb. Chem. 1, 184, (1999).-   17. T. Masquelin, Helv. Chem. Acta, 646, (1998).-   18. J. Heterocyclic Chem. 25, 959, (1988).-   19. a. Tet Lett. 38, 211, (1997)    -   b. J. Med. Chem. 39, 4156, (1996)    -   c. Synthesis, 147, (1986)-   20. Tet. Lett., 6557, (1998).-   21. Substitution of 4-sulfinyl derivative with OH will lead to    4-pyrimidone. J. Heterocyclic Chem. 22, 49, (1985).-   22. Katrritzky A., Tet. Lett., 39, 8051, (1998).    16.2 lndolo[2,3-b]quinoline 6,6,5,6 cyclic Scaffold

The indolo[2,3-b]quinoline 1a,b synthetic pathway is outlined in thepathway below. The key step in this synthesis is the decomposition ofthe corresponding triazoles 2a,b in polyphosphoric acid (PPA) at110-160° C., which affords the desired 1a,b^(1,2). The isomers 2a and 2bcan be separated during Purification. The starting triazoles 2a,b can beprepared by heating trisubstututed chloroquinolines 3 with benzotriazolebuilding blocks 6a,b at 110-120° C. in presence of TEA.^(1,3). Thebenzotriazole building blocks 6a,b is prepared from monosubstitutednitro-anilines by reduction of NO₂ group (SnCl₂ or H₂/Pd) and subsequentdiazotization of readily obtained diamines.^(1,4).

Synthesis of Indolo[2,3-b]quinoline

2-chloro-quinolines 3 is prepared in three steps from disubstitutedanilines first the anilide is formed either by reaction withpreactivated (BTC, DMAP, collidine) β-keto-acids, or with the free acidat high temperature followed by intramolecular cyclization of 5 underacidic conditions. Finally the obtained quinolinone is chlorinated withfreshly distilled POCl₃ ⁵ to afford 3. Another approach, namelysolid-phase synthesis of 1a,b, can be utilized using disubstitutedanilines with solid support attachable functional groups (CO₂H, NH₂,OH).

Solid Phase Synthesis of Indolo[2,3-b]quinoline

The starting anilines can be loaded on appropriate resin according tothe type of the functional group to be attached. If the functional groupis CO₂H, the resin will be phenolic (see quinoline chapter changeformulation according with the format of the patent) and the loading isperformed under esterification conditions (BTC, DMAP); if the functionalgroup is OH, the loading can be performed by Mitsunobu reaction; and ifthe functional group is NH₂ the starting aniline will be loaded undersulfonation conditions on sulfonyl chloride resin or alternativelyprepared by Curtius rearrangement from corresponding carboxylderivatives.

REFERENCES

-   1. Bioorg. Med. Chem., 7, 2457, 1999-   2. Arch Pharm 321463, 1988-   3. Tet Lett 39 1827 1998-   4. Org Syn Col Vol 1 3 106-   5. Org Syn Col Vol 3 194-   6. for other synthetic method for the preparation of    Indolo[2,3-b]quinoline see    -   a. from acylbenzotriazole and acyl isocyante, J Org Chem 65 8069        2000    -   b. coupling of 3-bromoquinoline with 2-amino boronate, Synlett        1067 1997    -   c. via a modified Graebe Ulmann reaction, J. Med Chem 37 3503        1994        16.3 Isoindoloindoles and Isoindoloindolones 6,5,5,6 Tetra        Cyclic Scaffolds

Herein, is described the Pd catalyzed annulation¹ to form anisoindoloindole skeleton from readily prepared imines and internal arylacetylenes.

Imines and disubstituted acetylenes undergo a multistep reaction in thepresence of palladium catalyst to produce isoindoloindoles², which areobtained in good yields (see pathway below).

General Scheme for the Preparation of Isoindoloindoles

By using divers building blocks—either mono- or di-substitutediodo-anilines 7, and premade di- or trisubstituted phenyl acetylenes 5.

A large library of isoindoloindols 1-4 can be obtained (see pathwaybelow).

This annulation reaction comprise of two steps synthesis withoutisolation of intermediate iodoimines 6. The steps of the synthesis areas follows:

1. Imine 6 is formed in solution using drying reagents such as TMOF,molecular sieves or Na₂SO₄.

2. The acetylenes 5 are prepared by Heck reaction between commerciallyavailable or pre-formed di- and monosubstituted iodobenzenes andmonosubstituted acetylenes using standard Pd catalysts³⁻⁸ (see pathwaybelow). Modified Heck reaction on solid phase can also be used⁹⁻¹². Whenwe use solution phase, the reaction mixture can be used for the nextstep as it, without recovering the catalyst, because the one is requiredfor the next step.

Heck Reaction for the Preparation of Substituted Phenyl Acetylenes

3. The annulation of internal alkynes to isoindoloindoles using Pd(OAc)₂in the presence of an anine LiCl or Bu₄NCl in DMF.

When one of the substituents is at ortho-position, the ring closure willproceed in regioselective manner affording single tetra-substitutedisondoloindoles 1,3.

When ortho-position on 5 is unoccupied, some substituents controlregioselectivity of ring closure by chelating the palladium in theσ-palladium intermediate, which is formed during the reaction. Othercases the two isomers can be separated by chromatography.

For generation of 11-hydroxy isoindoloindoles: TMS protectedhydroxyalkyne 11 can be utilized, generating after TMS removal (n-Bu₄NE)11-hydroxy-isoindoloindole sub-library 12 (see pathway below).

For generation of 11-amino-isoindoloindoles, carboxyalkyne 5 can be usedfor preparation of 11-carboxy-isoindoloindoles 14. The last can beconverted to corresponding azodocarbonyl 14 (n-BuOCOCl, then NaN₃)¹³⁻¹⁵(see pathway below), which can undergo rearrangement through nitreneintermediate to provide desired 11-amino-isoindoloindole sub-library 13.

Constant polar functional group can be added such as guanidine. The mostconvenient location for this purpose is the para position on phenyl ringderived from imine 10 (see pathway below). The imine 10 bears Bpocprotected amine group, which can readily be deprotected, afterannulation with appropriate alkyne, to give 9. Amino isoindoloindole 9can react with bis-Boc thiourea¹⁶ (HgCl₂, TEA) to obtain, aftersubsequent deprotection (TFA/DCM), the final library 8.

16.3.1 Isoindoloindolones

A slightly modified isoindoloindolone scaffold (see below) can beprepared by two systematic routes:

A schematic description is shown in the pathway below:

The approach presented above is divided to three major steps:

1. Formation of di- or tri-substituted indoles: via—Heck reactionbetween an acetylene and iodoaniline

2. Benzoylation of indole ring with ortho-iodo-benzoyl moiety. Thecoupling of disubstituted ortho-iodo benzoic acid BB to indole 18 can becarried out in to ways: 1. Regular coupling of BB to indole usingDCC/DMAP¹⁷; 2. Using a pre formed acid chloride^(18,19).

3. Cyclization using Pd catalyzed reaction (Heck annulation)^(20,21).The addition is very specific using iodo-benzoyl ring. In case the7^(th) position is not occupied it can add to 7 position of the indoleinstead of position 2. This addition gives us a new scaffold, which isanother library (see pathway below).

The indole 18 can be prepared by traceless solid phase indole synthesisusing indole N—H as a resin attachment point²², which could be cleavedto give the free indole 18. One of the most efficient solution phasemethods of indole synthesis is the Pd(0)-mediated reaction of2-iodo-anilines with acetylenes in the presence of base as developed byLarock^(23,24). Monosubstituted 2-Iodoaniline, after loading onto theTHP resin through an aminal linkage using PPTS can give 20 (see pathwaybelow). Replacing the catalyst to Pd(PPh₃)₂Cl₂ and using the DCE solublebase TGM, were found to be beneficial in pushing the annulation reactionto completion, affording 19. Resin cleavage with 10% TFA then can givethe free indole 18. It was found that TMS-substituted acetylenes readilywent to completion at 80° C. with almost complete regiocelectivity.

The carboxylated 15 (R₂═CO₂H) can be converted to amine analog 16through the corresponding azodocarbonyl, which can undergo rearrangementthrough nitrene intermediate to provide desired amino-isoindoloindolonesub-library.

Preparation of Isoindoloindolone

The hydroxy- and carboxy isoindolones 23 (X═O, CO₂) can be generated bySP synthesis (see pathway above) starting by loading the appropriateiodo-aniline on the resin 9 and effecting the annulation with TMSacetylenes.

The subsequent benzoylation and annulation of 27 followed by cleavagefrom the resin affords 25.

A second way of formation of isoindoloindolones presented in thefollowing pathway²⁵:

A key step is an intramolecular wittig reaction. Substituted ortho-alkylanilines and phthalic anhydride derivatives react to form arylphthalimides. These can be converted to phosphonium salts and can beclosed to isoindoloindolone system.

REFERENCES

-   1. Larock R. J. Am. Chem. Soc. 121, 3238, (1999).-   2. Roesch K. Org. Lett., 1551, (1999).-   3. Macdonald G. Chem. Commun. 2647, (1996).-   4. Amatore C. J. Org. Chem. 60, 6829, (1995).-   5. Amatore C. J. Org. Chem. 61, 8160, (1996).-   6. Lavastre O., Tetrahedron, 53, 7595, (1997).-   7. Cai M. Synthetic Commun. 27, 1935, (1997).-   8. Watanabe T. Syn Lett. 207, (1992).-   9. Collini M. Tet. Lett. 38, 7963, (1997).-   10. Tet. Lett. 38, 2307, (1997).-   11. Tet. Lett. 38, 2439, (1997).-   12. Amatore C. J. Org. Chem. 61, 5169, (1996).-   13. Rawal V. Tet. Lett. 35, 4947, (1994).-   14. Csuk R. Tet. Lett. 36, 7193, (1995).-   15. Paik S. Tet. Lett. 37, 5303, (1996).-   16. Atigada V. Bioorganic & Medicinal, Chemistry, 2487, (1999).-   17. Kraus G. Synthetic Commun. 23, 55, (1993).-   18. Kozikowski A. Tet. Lett. 32, 3317, (1991).-   19. Black D. Tetrahedron 49, 151, (1993).-   20. Shao H. Tet. Lett. 39, 7235, (1998).-   21. Desarbre E. Hetrocycles 41, 1987, (1995).-   22. Smith A. Tet. Lett. 39, 8317, (1998).-   23. Larocke R. J. Org. Chem. 60, 3270, (1995).-   24. Larocke R. J. Org. Chem. 63, 7652, (1998).-   25. J. Heterocycles Chem. 21, 623, (1984).    16.4 The Single Atom Scaffold

The smallest scaffold used in this implementation is the single atomscaffold, namely one carbon scaffold, of the general structure a:

The library a consists of several sub-libraries b-e (see below) thatrepresent compounds with one constant functional group and independentvariety of substituents around the carbon:

The secondary sub-libraries comprising two or three constant polarfunctionalities (see below) may be somewhat limited, because of thechemical unstability of molecules bearing two or three geminal amines orhydroxyl atoms (compounds f-j):

However, the synthesis of the α-amino acids k, α-hydroxy acids m andα-dicarboxylic 1 acids are known. For example they are described in:Synthesis of optically active α-amino acids by Robert M. Williams,Pergamon Press.

Some of the compounds based on the carbon scaffold are mostlycommercially available.

Those that are not commercially available can be synthesized, mostly insolution, by conventional methods.

The tetriary alcohols b¹ can be synthesized through the well knownepoxidation of olefins 2 (as a key step, producing epoxides 1, whichalready possess the required substituents² (see pathway below)

Electron-donating groups typically increase the rate. Conditions aremild and yields are high. The consequent reduction of epoxides is easilycarried out. The most common reagent is LiAlH₄, which reacts through theinversion of configuration 2³. As expected from the SN2 mechanism,cleavage usually occurs so that the desired tertiary alcohol b isformed. Product b serves as the starting material for the tertiaryamines c, which are obtained from b by substitution of correspondingtrifluoromethylsulfonate with ammonia in dioxane.

The solid phase preparation of the tertiary alcohols b has been recentlyreported⁴. Actually, this new cleavage strategy involves addition ofcarbon nucleophiles to ester bound polymers 3.

By this mode can be prepared only tetriary alcohols with two identicalalkyl or phenyl substituents (R2), thus, limiting the diversity of theproducts, but still able to generate rapidly the secondary sub-libraryof the tetriary alcohols.

The α-hydroxy acids m can be obtained by straightforward one-potprocedure from the corresponding α-keto acids 4 (pathway below). α oxoacid 4 are commercially available and their treatment with Grigniardreagents (2 equiv., THF, −40° C.-RT) lead to the desired m products.

A Schiff base activated glycine supported on a soluble polymer (PEG) 6can be readily alkylated with the wide variety of electrophiles in thepresence of carbonate base (Cs₂CO₃) in acetonitrile⁵ providingnon-stereospecific amino acid esters.

Similarly, Schiff base activated amino acids t-Butyl esters 8 can bealkylated to α-C disubstituted analogs 7 (pathway below) using alkylbromides and the LDA as a base (LDA, THF, −40° C.).

The Schiff bases 8 can be prepared by transimination of the commerciallyavailable t-Bu ester of amino acids 9 with benzophenone imine. Finally,The alkylated product can be totally deprotected by TFA/DCM yielding thedesired secondary sub-library k.

It should be mentioned that all products generated in this chapter areenantio-unselective and require separation of enantiomers on chiralcolumn. The utilization of racemic mixtures could be also considered

REFERENCES

-   1. Tetrahedron, 2855, (1976).-   2. Russ. Chem. Rew., 986, (1985).-   3. J. Org. Chem., 52, 14, (1981).-   4. S. Chandrasekhar, J. Comb. Chem., 2, 246, (2000).-   5. a. B. Sauvagnat, Tet. Lett., 39, 821, (1998).;    -   b. B. Sauvagnat, J. Comb. Chem., 2, 134, (2000).        16.5 Benzodiazepines 6,7 Bicyclic Scaffold

Benzodiazepines are therapeutic and anticonvulsant agents. As such the1,4 benzodiazepines have been the target of several solid phasesynthetic strategies.

The synthesis of 1,4-benzodiazepines, is based on the closure of a sevenmembered ring, via lactamization in high yield.¹⁻⁸

A slightly modified solid phase approach, which is based on the ringclosure, via an imine moiety is described in the pathway below.

Solid Phase Synthesis of Benzodiazepines

According to this strategy the aldehyde resin 1³ is coupled toβ-amino-alcohol 2 via reductive alkylation (FIG. 1). β-aminoalcohol (2)can be prepared in two alternative routes (see pathway below):

(1) Coupling of N-methoxyhydroxamate (8) with Griniard reagents (R₂MgBr)to obtain the corresponding ketones, followed by reduction using NaBH₄(MeOH, rt, few hours) to afford the Boc protected amino-alcoholderivative (9). Removal of the protecting group yield 2.

(2) Reducing N-methoxyhydroxamate (8) with LiAlH₄ to the aldehydederivative followed by coupling with Grinard reagents (R₂MgBr) to formthe Boc protected amino-alcohol derivative (9). Removal of theprotecting group yield 2.

Synthesis of β-aminoalcohol

The coupling between the aldehyde resin (1) and the amino-alcoholhydrochloride salt (2) is done via reductive alkylation usingNaBH(OAc)₃, 1% AcOH, DMF to give the resin immobilized β-amino-alcohols3. To avoid racemization, it is desirable to obtain equilibrium betweenthe resin bound aldehyde 1 and β-amino-alcohols 2 before addition of thereducing agent to the reaction mixture.

Coupling between the secondary amine 3 and Boc protected disubstitutedanthranilic acids 4 leads to resin bound intermediate 5. Oxidation ofthe hydroxy group to affords 6. The oxidation on solid support can becarried out by Py.SO₃ ⁹ complex in DMSO at room temperature, or by thealternative procedure using NMO¹⁰ (N-methylmorpholine N-oxide) with TPAP(tetra-n-propylammoniumperruthenate) catalyst, in DMF at roomtemperature.

Compound 6 is deprotected (TFA/DCM), and the free amine undergoesintramolecular cyclization under acidic conditions to obtain the desiredbenzodiazepine 7.

Introduction of amine or hydroxyl at position 3 of 1,4 benzodiazepineswill result in decomposition of the material. At position 2, an OH groupwill isomerise to the keto form, while an NH2 group can form tautomerswith the imine group.

The synthesic route for the preparation of a benzodiazepine having anNH₂ substituent at position 2 is described in the two pathways below:

(1) Thioamino ester (10) is loaded onto aldehyde resin 1 by reductivealkylation (NaBH(OAc)₃, 1% AcOH in DMF) to obtain resin boundintermediate 11 (FIG. 3). The secondary amines (11) is coupled withdisubstituted anthranilic acids (12) (EDC, NMP) to form amide 13, whichcan undergo the intramolecular cyclization using lithiated p-methoxyacetanilide (14)¹ to give thiobenzdiazepine 15. The cyclic resin boundthiointermediate 15 is submitted to methylation (MeI) followed byoxidation to generate preferable leaving group (namely methylsulfoxide)for nucleophylic substitution. Such substitution reactions can beoperated with acid labile dimethoxy benzylamine under standardconditions (16)(DMF, DIEA) providing after acidic cleavage the desired2-amine benzodiazepine sub-library 17.

(2) An alternative synthesis of 2-aminobenzodiazepine is as follows,Benzodiazepine 2,5 dione (20) is formed by coupling of substitutedanthranilic acid with amino-acid followed by ring closure, which reactswith Lawesson reagent to form intermediate-2-thiobenzodiazepine-5 one(21). The amine 22 is obtained by reaction between thebenzodiazepinethione 21 and ammonia.

Synthesis of 2-aminobenzodiazepine

Alternative Synthesis of 2-aminobenzodiazepine

The synthesis of β-hydroxy α amino-acid, a building block used for thepreparation of 2-carboxy benzodiazepine is described in the followingpathway. Commercially available chiral Fmoc serine t-butyl ester 26,undergoes Sworn oxidation ((COCl)₂, DMSO) to obtain the aldehyde 27. Thealdehyde 27 is subjected to Gringard reaction R1MgX to form the Fmocprotected amino-alcohols, which after Fmoc removal (piperidine, MeOH)leads to desired building blocks 28. In case when both R₁ and R₂ arecarboxyl groups, the starting material is di-t Butyl fumarate 23, whichupon epoxidation (mCPBA, NaHCO₃, DCM) gives the epoxide 24, followed byammonia in methanol to afford 25.

Preparation of b-hydroxy amino acid

The synthesis of benzopyridodiazepine^(11,12) 33 is described in thepathway below. 2-chloro-3-aminopyridines 29¹² is coupled withdisubstituted azidobenzoyl chloride building block 30. Reduction of theazide 31 with SnCl₂ provides the 2-chlorooxazolidine intermediate 32,which upon treatment with acid rearranges to the desired pyridine-basedtricyclic scaffold 33.

Preparation of Benzopyridodiazepine

The synthesis of the oxy analog of 33 namely10H-Dibenzo[b,f][1,4]oxazepin-11-one is described in the pathway below.Disubstituted O-aminophenol building unit 35 is attached to the resin onthe Acid sensitive MEthoxy BenzAldehyde (AMEBA)(34) via reductiveamination, to form 36.

Resin 36 was further modified with monosubstitued2-fluoro-5-nitrobenzoic acid 37 using HOAt/DIC strategy to affordimmobilized substrate 38, which was ready for the assembly of thenitro-10H dibenz[b,f][1,4]oxazepin-11-one analogs 39 (The keycyclization step (S_(N)Ar) between the fluor and the phenolic oxygen wasperformed using a 5% DBU in DMF^(23, 24, 25)). The reduction of thenitro group in the resulting resin can be obtained with the 1.5 Msolution of SnCl₂H₂O in DMF, and subsequent cleavage (TFA/DCM) from theresin 2-amino sub-library 39 is obtained.

Synthesis of dibenzo-oxazepinone REFERENCES

-   1. J. Org. Chem, 62, 1240, 1997,-   2. JCC, 2, 513, 2000,-   3. Synthetic Com., 21, 167, 1991-   4. J. Org. Chem, 60, 5742, 1995,-   5. Tet. Lett, 39, 7227, 1998-   6. J. Org. Chem, 63, 8021, 1998,-   7. Tet. Lett, 37, 8081, 1996;-   8. J. Org. Chem, 60, 5744, 1995.-   9. J. Am. Chem. Soc., 116, 2661, 1994-   10. J. Org. Chem, 61, 8765, 1996.-   11. J. Het. Chem., 23, 695, 1986-   12. J. Org. Chem, 62, 6102, 1997.-   13. Tet, 55, 2827, 1999;-   14. Tet, 55, 8295, 1999;-   15. Tet. Lett, 40, 5827, 1999    16.6 Pyrazinoquinazolinone-6,6,6 Tricyclic Scaffold

The pyrazino[2,1-b]quinazoline-3,6-dione system can be considered as aconstrained peptidomometic and is present in several families of naturalproducts. Some of these compounds exhibit very interesting biologicalactivity (J. Antibiotics 46, 380, 1996, Annu Rev Biochem 62 385, 1993).

One currently known syntheses of this scaffold can be grouped asfollows:

a: Transformation of 4-substituted 2,5-piperazinediones into thecorresponding iminoethers followed by cyclocondensation with anthranilicacid or methyl anthranilate.¹⁻⁵

Iminoether Anthranilic Acid Condensation to Pyrazinoquinazolinone

b: Acylation of 4-substituted 2,5-piperazinedione with o-azidobenzoylchloride followed by Staudinger reaction with phosphine to yield thecorresponding γ-phosphazene and subsequent intramolecular aza wittigcyclization of the latter imtermediate.^(6,7)

Pyrazinoquinazolinone Via N-o-azidobenzoyl-diketopiperazine

In a modified reaction sequence the N-o-azidobenzoyl-diketopiperazine isformed via an open chain tripeptide where the anthranilic acid unit isthe N terminal unit bears an azido group as masked amino function⁸.Cyclization generates the quinazolinone ring.

c: Double cyclization of an open chain tripeptide via4-imino-4-H-3,1-benzoxazine intermediate prepared throughcyclodehydration of a suitable o-acylanthranilamide in the presence ofiodine triphenyl phosphine.

This method was reported in solution⁹⁻¹³ as well as on solid phase¹⁴,which makes it a good mean for parralel array synthesis thereforesuitable for our purpose.

Pyrazinoquinazolinone Via Benzoxazine Intermediate

The tripeptide 6 is prepared by direct coupling of the amino acid esters(AA-OR) 3 with antranilic acid mediated by EDC. Condensation of 4 withthe Fmoc amino acid chloride 5 under two phase Scotten-Bauman condition(CH2Cl2, aq N2CO3) yields the tripeptide 6. amino acid chlorides 5 areprepared in situ by pre-activation of the corresponding Fmoc-AA-OH withBTC (triphosgene) and collidine in THF, DCM or Dioxane¹⁵. Theseconditions afford AA Clorides without racemization.

The transformation of the linear tri peptide to oxazine was accomplishedusing Wip's conditions (PPh₃/I₂/tertiary amine in large excess)Deprotection followed by rearrangement to quinaoline occurred upontreatment with 20% piperidine in methylene chloride. The cyclization toquinazoline is susceptible to steric hindrance and in case ofR3,R4=bulky groups cyclization requires stronger condition (DMAP refluxCH₃CN). Some epimerization (5%) took place in case in some of theexamples.

The application of the s synthesis in solution described above tocombinatorical synthesis on solid phase initiates with loading of Wangresin with appropriate amino acid (AA) affording 7. For majority of AAthe preloaded Wang resin is commercially available. 7 was deprotected(piperidine in DMF) and appropriate anthranilic acid along was coupled(EDC) to obtain 8 (pathway below).

SPS of Pyrazinoquinazolinone

The next step is acylation of aniline 7, with Fmoc-AA-Cl to obtainlinear tripeptide 9. The next step is the key dehydrative cyclization oflinear tripeptide 9 to 10. To ensure complete conversion, 10 equivalentsof Ph₃P were used. The final reaction is piperidine mediateddeprotection of Fmoc group and rearrangement of oxazine 10 to amidinecarboamide 11. After washing, the resin was refluxed in acetonitrile toinduce cyclative cleavage of 11 obtaining the desiredpyrazinoquinozaline library 1. The yields and purity of crude compoundswere claimed to be relatively high¹⁴. Final products 1 can be obtainedin few cases as mixture of cis:trans diasteridisomers (usually the ratiois 5-8:1). the larger degree of epimerization on solid phase is probablydue to the cyclizative cleavage, and HT purifier can separate theproducts. The above synthesis nicely illustrates the favorable featuresof the synthetic route. The first two steps involve peptidecouplings—the reaction for which SPPS was developed and which proceedsin almost quantitative yield for a variety of amino acids. Thedehydration of the liner tripeptide 9 requires large excess of Ph₃P,iodine and TEA—reagents which are readily removed by simple filtrationon solid phase. The ester functionality undergoing cyclization in thefinal step was chosen as the position for solid-phase attachment,resulting in self-cleavage from the resin.

The synthesis of pyrazinoquinazoline scaffold requires 3 building blocksthe 2 amino acids 3,5 and disubstituted anthranilic acid 2.

The amino acids and the Fmoc-amino acid are commercial available.

In order to introduce hetero functionalities (NH₂, OH) to Pyrazine ring(R3, R4) the synthesis of protected α-hydroxy-AA 14 and α-amino-AA and12 should be performed. AA 12 is known in literature¹⁶ and the synthesisis illustrated in the pathway below:

Synthesis of Protected α-amino-α-OH amino acids

Another AA 14 can be prepared by the similar mode through thecondensation between glyoxylic acid and FmocNH₂ in presence of t-BuOH inboiled toluene affording the desired 14.

Out of the third building block 3,5 dimethyl anthranilic acid iscommercial the other substituted anthranilic acid should be prepared ina tailor-made synthesis.

3-methyl-5-phenyl-anthranilic acid 15 can be prepared by bromination ofthe commercial available 3-methyl-anthranilic acid 16¹⁷. Followed bySuzuki reaction¹⁸.

Preparation of 3-methyl-5alkyl or phenyl anthranilic acid

3,5-diphenyl-anthranilic 17 acid will be prepared from the correspondingdibromoanthranilic acid 18 (commercial) via Pd catalyzed cross couplingreaction with excess of phenyl boronic acid¹⁹ (Aldrich).

Preparation of Diphenyanthranilic Acid

Substituted anthranilic acid can also be prepared from the correspondingsubstituted aniline 19 using a modified Sandmayer methodology. Reactionof the aniline with chloral and hydroxylamine affords theisonitrosoacetanilide followed by cyclization in sulphuric acid yieldsisatin 20. Oxidation of the later with H₂O₂ affords anthranilic acid ²⁰21. (see pathway below)

Preparation of Anthranilic Acids Via Isatin

Anthralinic acids substituted in position 3 with an OH group 22 can beprepared following the reaction sequence described in the pathway belowusing 4-substituted anilines (Et, Pr, Me Aldrich) as starting materials.The aniline was first brominated (23) followed by selectivelymonomethoxylation in the presence of CuI. The2-bromo-6-methoxy-4-alkylaniline 24 thus obtained was carbonylated usingPd complex as catalyst (CO, Pd(PPh₃)₂Cl₂) (=>25) and the final step isdeprotection by hydrolysis in concentrated hydrobromic acid²¹.

Preparation of 3-hydroxy-5-alkyl anthranilic acid

4-alkylaniline 19a can also serve as a starting material for thepreparation of dialkyl anthranilic acid 26 and 5-alkyl 3-phenylanthranilic acid 27 as described in the pathway below

3-alkyl-5-carboxylanthranilic acid 27 can be prepared starting fromo-alkylaniline 19b that is converted to isatin 20a (1. chloral, NH2OH,2. H2SO4), followed by bromination and oxidation to obtain the 5-bromoanthranilate 28 Substitution of the bromo with cyanide (29) andhydrolysis affords the 3-alkyl-5-carboxyl-anthranilic acid²² 27.

REFERENCES

-   1. Tetrahedron Asym 9 3025 1998-   2. Tetrahedron Asym 11, 3515, 2000-   3. Tetrahedron 55 14185 1999-   4. Tetrahedron 54, 969, 1998-   5. Tetahedron Asym 113515, 2000-   6. JACS 121 11953 1999-   7. Tetrahedron 57 3301, 2001-   8. JOC 65 1743 2000.-   9. JOC 63 2432 1998-   10. Tetrahedron Lett 40, 5429, 1999-   11. Org Lett 2, 3103, 2000-   12. JOC 65, 1022, 2000-   13. J. Org. Chem., 63, 2432, 1998-   14. J. Com. Chem., 2, 186, 2000-   15. a. J. Peptide Res., 3, 507, 1999. b. Tet Let,. 34 3861, 1993-   16. Proc. Natl. Acad. Sci. USA, 93, 2031, 1996-   17. Tet Lett. 41, 21083, 2000-   18. J. Am. Chem. Soc. 112, 2707, 2000-   19. synthesis 1410 1995-   20. a. Synth. Commun 29, 3627, 1999 b., J. Org. Chem 59, 6823,    1994, c. J. Med. Chem. 34, 1896, 1991, d. J. Indian. Chem. Soc. 66,    39, 1989, e. Tet Lett 29, 3709, 1988, f. J. Med. Chem 30, 1166, 1987-   21. J Med Chem 25 267 1990,-   22. Tetrahedron, 50, 2543, 1994    16.7. Pyrrole-5 Membered Ring Scaffold

In this chapter is described the comprehensive synthesis oftetra-substituted pyrroles. The proposed synthetic methods are on SolidPhase (SPS) as well as in solution.

Sub-library a which has a carboxyl group at position 2 is prepared insolution. The synthesis starts from nitrosation of β-keto esters toobtain oximes 3, which by reductive condensation with 1,3-diketones leadto ethyl carboxyketopyrrols 5¹ (pathway below). Pyrroles 5 undergoreduction of the carbonyl group to methylene¹, following by hydrolysisof ethyl carboxylate to afford the sub-library a. Curtius rearrangementmay convert the carboxyl into amine resulting in the conversion ofsub-library a to sub library b most conveniently. (in case R # R2mixtures of two isomers are obtained and may be separated).

Synthesis of Sub-Libraries a, b

Two building blocks are required for the synthesis of sub library a, andb, β-ketoesters, 1,3 diketones which are mostly commercially available.

Compounds of sub-library c can be obtained by the synthetic methoddescribed in the pathway below. In contrast to the former method thisapproach involves solid phase synthesis (SPS). Namely: condensation of1,2-diketones 7 with pre attached Boc imino diacetic acid mono ester 6as follows:

Synthesis of Additional Ten Compounds of Category a by SPS

The reaction^(2, 3) is performed under basic conditions using NaOMe orKOtBu. Imino diacetic acid 6 can be easily prepared from t-Bu ester ofGly by reductive amination of Glyoxylic acid using Sodiumcyanoborohydride as a reduction reagent and subsequent introduction of aBoc protecting group in multi-gram scale⁴.

Sub-library e can be prepared using the method described in the pathwaybelow. Position 3 in the resulting products has a fixed subtituent—anhydroxy group. Again, SPS is involved using pre-prepared building blocksas described above.

The process initiates from preparing five acyl Meldrumn's acid buildingblocks (12)^(5,6) in solution by reaction of acid chlorides 10 withMeldrum's acid 11 to give, in the presence of pyridine the correspondingcompound 12 almost quantitatively^(7, 8.)

Thus, heating 12 (5 equiv.) with the hydroxyl resin (the resin whichgenerates carboxylic acid, for example the oxime resin⁹) in THE atreflux for a few hours⁶ affords the polymer-bound β-ketoesters 13 withconcomitant release of CO₂ and acetone, which helps to drive thereaction to completion. The reaction could be easily monitored by FT-IRon the resin (KBr pellets). The fictionalization of the α-carbon of 13is performed with excess of the alkylating reagent, avoidingO-alkylation as well as double alkylation.

Thus, haloalkanes (36 equiv.) in the presence of 1 M TBAF⁸ in THF (26equiv., 3 h) easily convert 13 to 14 at RT (FIG. 4). Typically it isimportant to exclude traces of water, which may decrease the yield.Addition of an excess of presynthesized amino ketones 15^(10,11) (FIG.5) (20 equiv., 3 h, RT), to the resin linked p-ketoesters 14 inTHF/trimethylorthoformate (1/1) gives the Shiff bases 16, Cyclization of16 under basic conditions with concomitant release of the product 17into the solution followed by reduction of the ketone (R3=Me,Et). (NaBH4BF3OEt2)¹ produces sub library e.

The reaction can also be performed in solution using α-substitutedβ-ketoesters following the same reaction sequence.

It should be noted that β-hydroxy pyrroles may exist to some extent inits keto tautomer¹⁴ The required building blocks are β-keto esters whichare commercial or the α-substituted -β-ketoesters.

The α-aminoketone building block can be prepared from the correspondingamino acid hydroxamate as described in the following pathway.

Synthesis of Amino Ketones from Gly Boc Hydroxamates

N-protected glycine recats with N-O-dimethyl hydroxylamine to givehydroxamte 18 Reaction of the glycine hydroxamate with Gringard reagent(EtMgBr, MeMgBr) affords the ketone 19 no over adding is observed.Deprotection of 19 gives the amino ketone building blocks.

In case R3=OH, glycinate reacts with the substituted β-keto esters

Sixteen more products can be obtained by the method described in thepathway below. A key step for the preparation of sub library f isMichael addition of amino ketones 21 to DTAD (21)¹². The obtainedaminoolefine 23 undergoes cyclization in acidic conditions, to affordthe sub-library f.

Synthesis of 2-carboxy-3-amino-pyrroles

The synthesis of 2-carboxy, 3-amino pyrroles 28 is well known^(12,13)(see pathway above). It is performed through the enamine formation of 26and subsequent intramolecular cyclization of 27 under basic conditions(NaOEt) to give 28. The β-keto nitrites 25. can be prepared by eitheralkylation of 25¹⁵ or acylation of the corresponding nitrile.¹⁶

REFERENCES

-   1) J. Paine III, J. Org. Chem., 3857, (1976).-   2) M. Friedman, J. Org. Chem., 859, (1965).-   3) K. Dimroth, Ann. Chem., 639, 102, (1961).-   4) G. Byk, J. Org. Chem., 5687, (1992).-   5) L. Tietze, Bioorg. & Med. Chem. Lett., 1303, (1997).-   6) L. Tietze, SYNLETT, 667, (1996).-   7) Y. Oikawa, J. Org. Chem., 2087, (1978).-   8) L. Weber, SYNLETT, 1156, (1998).-   9) The Combinatorial Index, p. 15-   10) S. Nahm, Tet. Lett., 3815, (1981).-   11) Eur. J. Org. Chem 2809, 2000-   12) H. Ward, Tet. Lett., 25, 527, (1969).-   13) Mu-III Lim, J. Org. Chem., 3826, (1979).-   14) Aust J. Chem 20, 935, 1967.-   15) J Org Chem 55 429 1990-   16) a. Bull Chem Soc Jpn 62 3851 1989, b. Chem Pharm Bull 46 69    1998, c. J Med Chem 34 1741 1991    16.8 Thiophenes and Related Scaffolds

The chemistry of 2-aminothiophenes and related scaffolds has attractedspecial attention in the last 30 years because of their applications inpharmaceuticals, agriculture, pesticides and dyes.

The chemistry of 2-aminothiophenes are conveniently available throughthe, synthetic method developed by Gewald^(lab) who devised the mostfacile and promising synthetic route leading to 2-aminothiophenes A withelectron withdrawing substituents such as cyano, carbethoxy etc. in the3-positions and alkyl, aryl, cycloalkyl, and hetaryl groups in the 4-and 5-positions.

Gewald Reaction

The simplest version of the Gewald reaction consists of a one-potprocedure, namely condensation of aldehydes, ketones or 1,3-dicarbonylcompounds with activated nitrites and sulfur in the presence of amine atroom temperature. Ethanol, DMF, dioxane are preferred solvents andamines like diethylamine, morpholine, or triethylamine have beenused^(1-7.) This method offers considerable improvement over othermethods by replacing an a-mercaptoaldehyde or an a-mercaptoketone bysimpler starting materials. It is necessary to use 0.5-1 molarequivalents of amine based on the amount of nitrile to obtain highyield.

In another synthesis version a two-step procedure is preferred. Ana,b-unsaturated nitrile is first prepared by a Knoevenagle-Copecondensation and then treated with sulfur and an amine.

This two-step version of the Gewald reaction gives higher yields. Alkylaryl ketones do not give thiophenes in the one-pot modification, butgives acceptable yields in the two-step technique² (see pathway below).

Two Step Gewald Reaction

The use of t-butyl cyanoacetate instead of the ethyl ester enables toobtain free acid of 3-carboxy-2-aminothiophenes by convenient TFA/DCMhydrolysis⁸

The amino acid obtained as well as the protected acid can be used asbuilding blocks for further transformation to more complex scaffolds asis exemplified below:16.8.1 5,5 Bicyclic Scaffolds

Thienopyrrole Synthesis

Thienopyrrole scaffold B⁹ (pathway above) is prepared by the reaction ofaminocarboxylate A with bromoacetate (K₂CO₃) to obtain diesterintermediate 1, which after acetylation (compound 2) (30% AcCl in AcOH)undergoes Dieckmann condensation (EtONa, EtOH) to afford3-hydroxy-2-carboxy thieno[2,3-b]pyrrole B1. The amino analog B2requires starting with the 2-amino-3-cyano thiophene A1. Acetylationfollowed by alkylation with a-bromoacetate (K2CO3 acetone or NaH DMF)leads under similar reaction conditions to ring closure producing3-amino-caboxy thienopyrrole B2. Acetylation of the amine at position 2and LiOH are required to increase the nucleophilicity of the amine.

16.8.2 5,6-bicyclic Scaffolds

The thienopyridine scaffold C is prepared via modified Friedlanderreaction, namely reaction of thiophene A, A1 and 5 with b-ketoesters,1,3 diketones under basic condition to form thienopyridines as describedin the pathway below

Thienopyridine Synthesis

Another 5,6-bicyclic ring system—the thieno pyrimidine D is prepared bythe reaction of thiophene A, A1 with chloro formamidine hydrochloride 4,11

Thienopyrimidine Synthesis

16.8.3 5,8,5 5,8,6 Tricyclic and 5,5,8,6 5,5,8,5 Tetracyclic Scaffolds

The scaffolds E,F G and H can be generated from thiophenes are describedin scheme 6. These compounds result from the formation of an eightmembered ring Dilactam.

Preparation of 8 Membered Ring Dilactam

The formation of the eight membered ring includes several steps:

1. Activation of the β-amino acid using SOCl2^(12a) or POCl3^(12b) (inthese cases the amine should be protected by Boc) or by DCC^(12c) andmethyl chloroformate^(12d)

2. Coupling of the activated acid and another N-protectedβ-amino-t-butyl ester^(13,)

3. Deprotection of the t-butyl ester and the N-Boc amine using TFA inDCM

4. Coupling by PyBop or any other analog in case R′ is a benzyl group itcan be removed at this stage by hydrogenation.

16.8.4 5,7 Bicyclic Scaffold

The synthesis of I, J analogs of the benzodiazepines scaffold isillustrated in the following pathway. In both approaches chiral aminoacid are introduced into the synthesis raising the diversity around thea carbon. Thieno diazepine I is prepared from 2-amino-3-acyl)-thiophenes5. which reacts with pre formed Boc amino acid chloride (amino acid,BTC, collidine, THF or DCM). Deprotection of 8 (4N HCl) with concomitantring closure leads to 2-oxothienodiazepine I. Thiophenodiazepine J canbe prepared starting from 2-amino-3-carboxy-thiophenes A, which afterpre activation to the thienooxzaine dione (BTC, collidine, THF or DCM))reacts with amino ketone to obtain 9, ring closure afford5-oxothienodiazepine.

Synthesis of Thienodiazepines

The synthesis of Thienodiaepine K

The synthesis of thienodiazepine K is described in the above pathway.2-amino-3-acyl thiophene 5 is first acetylated with the appropriateα-haloacetyl chloride Nucleophilic substitution with NaI followed byammonia to obtain the amino amide¹⁵ 11. The latter undergoes ringclosure to the thienodiazepine K under acidic conditions Anotheralternative is to react thiophene 5 with the phthalide protected aminoacyl chloride, Deprotection with hydrazine (11) and ring closure toobtain thienodiazepine K¹⁶

The synthesis of thienozepine L is based on coupling of sucssinicanhydride or acid chloride monoester with thiophene 5 (see pathwaybelow) The obtained amide 12 undergoes intramolecular condensation (NaH)to provide the targeted compounds¹⁷.

Preparation of Thienozepine

Scaffold M, having a thienodiazepinone skeleton may be prepared asdescribed in the pathway below. The N-protected aminocarboxythiophene Ais first preactivated (BTC, collidine, DCM) and submitted to reactionwith α-amino acetonitriles 14 to afford amide 13.

The latter reacts under basic conditions (NaOMe) to provide through theintramolecular cyclization the disubstituted intermediate2-aminothieno-1,4-diazepin-5-one 15¹⁸. In the next step2-aminothieno-1,4-diazepin-5-one 15 is heated with acetyl hydrazineleading to thienotriazolodiazepinone M

The Synthesis of Thienotriazolodiazepinone

16.8.5 5,6,5,6 Tetracyclic and 5,6,5 Tricyclic Scaffolds

The thiophene substituted in the 3 position with a benzimidazole namelybenzimidazoloaminothiophene 16 can serve as a building block for thesynthesis of thieno(2′,3′,4,5)pyrimidino(1,6)benzimidazole,N N1, Thestarting material 2-cyanomethylbenzimidazole 16, is prepared fromsubstituted phenylene diamine 17 and malononitrile¹⁹Nitrile 18 issubmitted to Gewald reaction using elemental sulfur powder and ketones²⁰or cyanoacetamide²¹ in dry DMF containing a catalytic amount of TEAunder reflux to form thiophene 16 (see pathway below).

The Synthesis of Thienopyrimidinobenzimidazole

Condensation of 16 with aldehydes or ketones, afford N and N1respectively^(21,22).

The Synthesis of Thienopyrimidinodihydroimidazole

Using the same approach dihydroimidazoylacetonitrile 20²³ (see pathwayabove) and thienoimidazoyl-acetonitrile 21 (see pathway below) can beprepared from the corresponding diamines (ethylene diamine and thiophene2,3 diamine²⁴) and malonolitrile The resulting nitrites react withketones under Gewald conditions forming O, O1 and P, P1.

The Synthesis of Scaffolds P and P1

16.8.6 5-6-5-6 Tetracyclic Scaffold

Synthesis of Scaffold Q

4H-thieno[2′,3′:4,5]pyrimido[2,1-b]benzothia-or--zoles Q can be preparedfrom amino thiophene A as outlined in the pathway above.²⁵2-Amino-3-carboxythiophene undergoes condensation at high temperaturewith chlorobenzimidazole²⁶. chlorobenzthiazole 23 leading to thecorresponding thienopyrimidinazoles Q.

16.8.7 5-6-5 Tricyclic Scaffold

Thia-triaza-s-indacenone R (see pathway below), can be obtainedaccording to literature procedures In this synthesis the aminothiopheneA undergoes cyclization in boiling acetic acid with pre formedmethylthio imidazoles 24 to give the desired system. R

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It will be appreciated that the above described methods of targetmeasurement and drug discovery may be varied in many ways, including,changing the order of steps, which steps are performed on-line and whichsteps are performed off-line. In addition, various parallel and/orsequential configurations may be used to implement the above invention,optionally utilizing a variety of software tools and/or varioushardware/software combinations. In addition, a multiplicity of variousfeatures, both of methods and of devices has been described. It shouldbe appreciated that different features may be combined in differentways. In particular, not all the features shown above in a particularembodiment are necessary in every similar exemplary embodiment of theinvention. Further, combinations of the above features are alsoconsidered to be within the scope of some exemplary embodiments of theinvention. Also within the scope of the invention are computer readablemedia on which software, for performing part or all of an exemplaryembodiment of the invention, are written. It should also be appreciatedthat many of the embodiments are described only as methods or only asapparatus. The scope of the invention also covers hardware and/orsoftware adapted and/or designed and/or programmed to carry out themethod type embodiments. In addition, the scope of the inventionincludes methods of using, constructing, calibrating and/or maintainingthe apparatus described herein. Headers, where they appear, are providedfor ease of browsing and should not be construed as necessarily limitingthe contents of the section to that which is suggested by the heading.When used in the following claims, the terms “comprises”, “comprising”,“includes”, “including”, “having” or their conjugates mean “includingbut not limited to”.

It will be appreciated by a person skilled in the art that the presentinvention is not limited by what has thus far been described. Rather,the scope of the present invention is limited only by the followingclaims.

1. A method of obtaining information about a chemically active area of atarget molecule, comprising: providing a set of substantially rigidchemical gauges; causing said target to interact with a plurality ofgauges of said set of gauges; assaying said interaction of said gaugeswith said target to obtain a plurality of assay results; and analyzingsaid assay results to obtain information about said chemically activearea.
 2. A method according to claim 1, wherein said gauges allowrotation of moieties of said gauges.
 3. A method according to claim 1,wherein said gauges are constructed using a rigid scaffold.
 4. A methodaccording to claim 1, wherein constituent atoms of said gauges do notmove more than 1 Å unless at least 20 Kcal/Mol are applied to the gauge.5. A method according to claim 1, wherein analyzing comprisesidentifying a plurality of spatial and chemically specific bindingsconfigurations in said target active area.
 6. A method according toclaim 5, wherein said configurations comprise triangular configurations.7. A method according to claim 5, wherein identifying comprisesidentifying a configuration that matches a configuration of a boundgauge.
 8. A method according to claim 5, wherein identifying comprisesidentifying a configuration that does not match a configuration of abound gauge.
 9. A method according to claim 8, wherein identifyingcomprises identifying by statistical analysis of said assay results. 10.A method according to claim 9, wherein identify comprises identifying byclustering.
 11. A method according to claim 5, wherein identifyingcomprises assuming each gauge indicates a single configuration.
 12. Amethod according to claim 5, wherein identifying comprises assuming atleast some of the gauges indicate a plurality of configurations.
 13. Amethod according to claim 5, wherein identifying comprises classifyinggauges by chemical moieties at vertexes of said configurations.
 14. Amethod according to claim 1, comprising reconstructing a spatial map ofat least part of said chemically active area, from at least two of saidassay results, said part including at least four chemical binding areas.15. A method according to claim 14, wherein said part includes at leastsix chemical binding areas.
 16. A method according to claim 5,comprising reconstructing a spatial map of at least part of saidchemically active area, from at least two of configurations, said partincluding at least four chemical binding points.
 17. A method accordingto claim 16, wherein said part includes at least six chemical bindingareas.
 18. A method according to claim 16, wherein reconstructingcomprises: test-reconstructing a plurality of spatial maps from saidconfigurations; scoring said maps; and selected a spatial map based onits score.
 19. A method according to claim 16, wherein reconstructingcomprises: test-reconstructing a plurality of spatial maps from saidconfigurations; clustering said maps according to common substructures;and selected a spatial map based on a relative property of a cluster itbelongs to.
 20. A method according to claim 19, wherein said relativeproperty comprises size.
 21. A method according to claim 16, whereinsaid spatial map includes enough binding points to ensure binding of asmall molecule drug having a chemical profile matching the bindingpoints.
 22. A method according to claim 21, wherein said spatial mapincludes at least 6 binding points.
 23. A method according to claim 21,wherein said spatial map includes at least 8 binding points.
 24. Amethod according to claim 1, wherein said set of gauges comprises a setof gauges with at least 10,000 gauges.
 25. A method according to claim1, wherein said set of gauges comprises a set of gauges with at least50,000 gauges.
 26. A method according to claim 1, wherein said gaugescomprise moieties arranged in spatial configurations and wherein saidgauges are selected to span a virtual space of spatial chemicalconfigurations.
 27. A method according to claim 1, wherein substantiallyeach point of virtual space that is spanned by said gauges is covered byat least two gauges.
 28. A method according to claim 1, whereinsubstantially each point of virtual space that is spanned by said gaugesis covered by at least three gauges.
 29. A method according to claim 1,wherein at least 0.5% of said gauges bind with said target.
 30. A methodaccording to claim 1, wherein at least 1% of said gauges bind with saidtarget.
 31. A method according to claim 1, wherein at least 3% of saidgauges bind with said target.
 32. A method according to claim 1, whereinat least 50% of said gauges are defined by adding moieties to a set offewer than 100 scaffolds.
 33. A method according to claim 1, wherein atleast 50% of said gauges are defined by adding moieties to a set ofFewer than 50 scaffolds.
 34. A method according to claim 1, wherein atleast said set of gauges uses fewer than 15 different chemical moietiesto define the chemical behavior of said gauges.
 35. A method accordingto claim 1, wherein at least said set of gauges uses fewer than 10different chemical moieties to define the chemical behavior of saidgauges.
 36. A method according to claim 1, wherein said assay is afunctional assay.
 37. A method according to claim 1, wherein said assayis a binding assay.
 38. A method according to claim 1, wherein saidassay is a cellular assay.
 39. A method according to claim 1, whereinsaid assay is a flow-through assay.
 40. A method according to claim 36,wherein said functional assay is performed in the presence of a naturalsubstrate of said target.
 41. A method according to claim 1, whereinsaid target comprises a protein including a biochemically active areaadapted to engage a substrate.
 42. A method according to claim 41,wherein said chemically active area comprises an area including saidbiochemically active area.
 43. A method according to claim 41, whereinsaid chemically active area comprises a control area of said protein.44. A method according to claim 1, analyzing comprises analyzingsuccessful binding of at least 60 gauges.
 45. A method according toclaim 1, analyzing comprises analyzing successful binding of at least 10gauges.
 46. A method according to claim 1, analyzing comprises analyzingsuccessful binding of at least 100 gauges.
 47. A method according toclaim 5, wherein identifying comprises identifying at least 40 differentconfigurations.
 48. A method according to claim 5, wherein identifyingcomprises identifying at least 10 different configurations.
 49. A methodaccording to claim 5, wherein identifying comprises identifying at least100 different configurations.
 50. A method according to claim 16,comprising: comparing said map to a lead data base; and selecting a leadfrom said data base for further use responsive to a semblance or lack ofsemblance between said lead and said map.
 51. A method according toclaim 16, comprising: comparing said map to a lead data base; andrejecting a lead from said data base for further use responsive to asemblance between said lead and said map.
 52. A method according toclaim 16, comprising: constructing a lead to have a semblance to saidmap.
 53. A method according to claim 52, wherein constructing comprisesconstructing using said gauges or scaffolds used to define said gauges.54. A method according to claim 5, comprising: comparing saidconfigurations to a lead data base; and selecting a lead from said database for farther use responsive to a matching of said configurations tosaid lead.
 55. A method according to claim 5, comprising: constructing alead based on said configurations.
 56. A method according to claim 5,comprising: selecting at least one of said gauges as a lead for drugdiscovery.
 57. A method according to claim 1, comprising comparing thebinding of gauges with similar binding geometries to obtain stericclashing data; and analyzing said steric clashing data to providegeometrical information about said target. 58-101. (canceled)
 102. Amethod according to claim 1, comprising generating a set of drug leadsfor said target based on said information.
 103. A method according toclaim 102, comprising removing known drug leads for said target fromsaid set 104-154. (canceled)
 155. A method according to claim 1, whereinsaid analyzing comprises characterizing said chemically active area.156. A method according to claim 155, wherein said chemically activearea comprises at least two disjoint chemically active areas.
 157. Amethod according to claim 1, wherein said analyzing comprises takingsaid rigidity into account of said analyzing.
 158. A method according toclaim 1, wherein said target molecule comprises an agricultural chemicaltarget.
 159. A method according to claim 1, wherein said target moleculecomprise a drug target.
 160. A method according to claim 1, wherein, onthe averages each point of virtual space that is spanned by said gaugesis covered by between 1.1 and 2 gauges.
 161. A method according to claim1, wherein at least 0.1% of said gauges bind with said target.
 162. Amethod according to claim 1, wherein the moieties comprise Hydroxyl(OH), Carboxyl (COOH), Amide (CONH2). Ethyl (CH2-CH3), Propyl(CH2-CH2-CH3), Phenyl (C6H5, 6 member aromatic ring).
 163. A methodaccording to claim 1, wherein said chemically active area comprises atleast two disjoint chemically active areas.
 164. A method of mapping anactive area of a biological target, comprising: providing a set ofchemical gauge molecules, each gauge molecule having a geometricstructure; assaying an interaction between a plurality of said chemicalgauge molecules and the biological target; and mapping the active areaaccording to the results of assaying said interactions and according tosaid gauge molecule geometric structures.
 165. A method according toclaim 164, wherein said gauge molecules are rigid molecules.
 166. Amethod according to claim 164, in which the gauge molecules are composedof a rigid scaffold and to which various chemical moieties are attachedusing one rotationally free bond.
 167. A method according to claim 166,wherein the rigid scaffold containing no rotationally free bonds